Dehydrophenylahistins and analogs thereof and the synthesis of dehydrophenylahistins and analogs thereof

ABSTRACT

Compounds represented by the following structure (I) are disclosed:  
                 
 
as are methods for making such compounds, wherein said methods comprise reacting a diacyldiketopiperazine with a first aldehyde to produce an intermediate compound; and reacting the intermediate compound with a second aldehyde to produce the class of compounds with the generic structure, where the first aldehyde and the second aldehydes are selected from the group consisting of an oxazolecarboxaldeyhyde, imidazolecarboxaldehyde, a benzaldehyde, imidazolecarboxaldehyde derivatives, and benzaldehyde derivatives, thereby forming the above compound wherein R 1 , R 1 ′, R 1 ″, R 2 , R 3 , R 4 , R 5 , and R 6 , X 1  and X 2 , Y, Z, Z 1 , Z 2 , Z 3 , and Z 4  may each be separately defined in a manner consistent with the accompanying description. Compositions and methods for treating cancer and fungal infection are also disclosed.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.60/450,063 filed Feb. 23, 2003, to U.S. Provisional Application No.60/411,128 filed Sep. 16, 2002, and to U.S. Provisional Application No.60/401,074 filed Aug. 2, 2002. Each of those applications areincorporated herein by reference in their entireties. This applicationis also related to U.S. application Ser. No. ______, filed on this dateherewith, which is also incorporated herein by reference in itsentirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to compounds and methods of syntheticpreparation in the fields of chemistry and medicine. More specifically,the present invention relates to compounds and procedures for makingcompounds useful in the treatment of cancer and the treatment of fungalinfections.

2. Brief Description of the Related Art

It is thought that a single, universal cellular mechanism controls theregulation of the eukaryotic cell cycle process. See, e.g., Hartwell, L.H. et al., Science (1989), 246: 629-34. It is also known that when anabnormality arises in the control mechanism of the cell cycle, cancer oran immune disorder may occur. Accordingly, as is also known, antitumoragents and immune suppressors may be among the substances that regulatethe cell cycle. Thus, new methods for producing eukaryotic cell cycleinhibitors are needed as antitumor and immune-enhancing compounds, andshould be useful in the treatment of human cancer as chemotherapeutic,anti-tumor agents. See, e.g., Roberge, M. et al., Cancer Res. (1994),54, 6115-21.

Fungi, especially pathogenic fungi and related infections, represent anincreasing clinical challenge. Existing antifungal agents are of limitedefficacy and toxicity, and the development and/or discovery of strainsof pathogenic fungi that are resistant to drugs currently available orunder development. By way of example, fungi that are pathogenic inhumans include among others Candida spp. including C. albicans, C.tropicalis, C. kefyr, C. krusei and C. galbrata; Aspergillus spp.including A. fumigatus and A. flavus; Cryptococcus neoformans;Blastomyces spp. including Blastomyces dermatitidis; Pneumocystiscarinii; Coccidioides immitis; Basidiobolus ranarum; Conidiobolus spp.;Histoplasma capsulatum; Rhizopus spp. including R. oryzae and R.microsporus; Cunninghamella spp.; Rhizomucor spp.; Paracoccidioidesbrasiliensis; Pseudallescheria boydii; Rhinosporidium seeberi; andSporothrix schenckii (Kwon-Chung, K. J. & Bennett, J. E. 1992 MedicalMycology, Lea and Febiger, Malvern, Pa.).

Recently, it has been reported that tryprostatins A and B (which arediketopiperazines consisting of proline and isoprenylated tryptophanresidues), and five other structurally-related diketopiperazines,inhibited cell cycle progression in the M phase, see Cui, C. et al.,1996 J Antibiotics 49:527-33; Cui, C. et al. 1996 J Antibiotics49:534-40, and that these compounds also affect the microtubuleassembly, see Usui, T. et al. 1998 Biochem J 333:543-48; Kondon, M. etal. 1998 J Antibiotics 51:801-04. Furthermore, natural and syntheticcompounds have been reported to inhibit mitosis, thus inhibit theeukaryotic cell cycle, by binding to the colchicine binding-site(CLC-site) on tubulin, which is a macromolecule that consists of two 50kDa subunits (α- and β-tubulin) and is the major constituent ofmicrotubules. See, e.g., Iwasaki, S., 1993 Med Res Rev 13:183-198;Hamel, E. 1996 Med Res Rev 16:207-31; Weisenberg, R. C. et al., 1969Biochemistry 7:4466-79. Microtubules are thought to be involved inseveral essential cell functions, such as axonal transport, cellmotility and determination of cell morphology. Therefore, inhibitors ofmicrotubule function may have broad biological activity, and beapplicable to medicinal and agrochemical purposes. It is also possiblethat colchicine (CLC)-site ligands such as CLC, steganacin, see Kupchan,S. M. et al., 1973 J Am Chem Soc 95:1335-36, podophyllotoxin, seeSackett, D. L., 1993 Pharmacol Ther 59:163-228, and combretastatins, seePettit, G. R. et al., 1995 J Med Chem 38:166-67, may prove to bevaluable as eukaryotic cell cycle inhibitors and, thus, may be useful aschemotherapeutic agents.

Although diketopiperazine-type metabolites have been isolated fromvarious fungi as mycotoxins, see Horak R. M. et al., 1981 JCS Chem Comm1265-67; Ali M. et al., 1898 Toxicology Letters 48:235-41, or assecondary metabolites, see Smedsgaard J. et al., 1996 J Microbiol Meth25:5-17, little is known about the specific structure of thediketopiperazine-type metabolites or their derivatives and theirantitumor activity, particularly in vivo. Not only have these compoundsbeen isolated as mycotoxins, the chemical synthesis of one type ofdiketopiperazine-type metabolite, phenylahistin, has been described byHayashi et al. in J. Org. Chem. (2000) 65, page 8402. In the art, onesuch diketopiperazine-type metabolite derivative, dehydrophenylahistin,has been prepared by enzymatic dehydrogenation of its parentphenylahistin. With the incidences of cancer on the rise, there exists aparticular need for chemically producing a class of substantiallypurified diketopiperazine-type metabolite-derivatives having animalcell-specific proliferation-inhibiting activity and high antitumoractivity and selectivity. There is therefore a particular need for anefficient method of synthetically producing substantially purified, andstructurally and biologically characterized diketopiperazine-typemetabolite-derivatives.

Also, PCT Publication WO/0153290 (Jul. 26, 2001) describes anon-synthetic method of producing dehydrophenylahistin by exposingphenylahistin or a particular phenylahistin analog to a dehydrogenaseobtained from Streptomyces albulus.

SUMMARY OF THE INVENTION

Compounds, and methods for the synthetic manufacture of compounds, aredisclosed for a class of compounds having the structure of Formula (I):

The disclosed compounds have the structure of Formula (I) wherein:

-   -   R₁, R₄, and R₆, are each separately selected from the group        consisting of a hydrogen atom, a halogen atom, and saturated        C₁-C₂₄ alkyl, unsaturated C₁-C₂₄ alkenyl, cycloalkyl,        cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl,        heteroaryl, substituted heteroaryl, amino, substituted amino,        nitro, azido, substituted nitro, phenyl, and substituted phenyl        groups, hydroxy, carboxy, —CO—O—R₇, cyano, alkylthio,        halogenated alkyl including polyhalogenated alkyl, halogenated        carbonyl, and carbonyl —CCO—R₇, wherein R₇ is selected from a        hydrogen atom, a halogen atom, and saturated C₁-C₂₄ alkyl,        unsaturated C₁-C₂₄ alkenyl, cycloalkyl, cycloalkenyl, alkoxy,        cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted        heteroaryl, amino, substituted amino, nitro, azido, substituted        nitro, phenyl, and substituted phenyl groups;    -   R₁′ and R₁″ are independently selected from the group consisting        of a hydrogen atom, a halogen atom, and saturated C₁-C₂₄ alkyl,        unsaturated C₁-C₂₄ alkenyl, cycloalkyl, cycloalkenyl, alkoxy,        cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted        heteroaryl, amino, substituted amino, nitro, azido, substituted        nitro, phenyl, and substituted phenyl groups, hydroxy, carboxy,        —CO—O—R₇, cyano, alkylthio, halogenated alkyl including        polyhalogenated alkyl, halogenated carbonyl, and carbonyl        —CCO—R₇, wherein R₇ is selected from a hydrogen atom, a halogen        atom, and saturated C₁-C₂₄ alkyl, unsaturated C₁-C₂₄ alkenyl,        cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted        aryl, heteroaryl, substituted heteroaryl, amino, substituted        amino, nitro, azido, substituted nitro, phenyl, and substituted        phenyl groups;    -   R₂, R₃, and R₅ are each separately selected from the group        consisting of a hydrogen atom, a halogen atom, and saturated        C₁-C₁₂ alkyl, unsaturated C₁-C₁₂ alkenyl, acyl, cycloalkyl,        alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl,        substituted heteroaryl, amino, substituted amino, nitro, and        substituted nitro groups, sulfonyl and substituted sulfonyl        groups;    -   X₁ and X₂ are separately selected from the group consisting of        an oxygen atom, a nitrogen atom, and a sulfur atom, each either        unsubstituted or substituted with a R₅ group, as defined above;    -   Y is selected from the group consisting of a nitrogen atom, a        substituted nitrogen atom with a R₅ group from above, an oxygen        atom, a sulfur atom, a oxidized sulfur atom, a methylene group        and a substituted methylene group;    -   n is an integer equal to zero, one or two;    -   Z, for each separate n, if non-zero, and Z₁, Z₂, Z₃ and Z₄ are        each separately selected from a carbon atom, a sulfur atom, a        nitrogen atom or an oxygen atom; and    -   the dashed bonds may be either single or double bonds;    -   with the proviso that, in a particular compound, if R₁, R₁′, R₂,        R₃, R₄ and R₅ are each a hydrogen atom, then it is not true that        X₁ and X₂ are each an oxygen atom and R₆ is either        3,3-dimethylbutyl-1-ene or a hydrogen atom.

The methods comprise the steps of:

-   -   reacting a diacyldiketopiperazine with a first aldehyde to        produce an intermediate compound; and    -   reacting said intermediate compound with a second aldehyde to        produce said class of compounds with said generic structure,        wherein    -   said first aldehyde and said second aldehydes are selected from        the group consisting of an oxazolecarboxaldeyhyde,        imidazolecarboxaldehyde, a benzaldehyde, imidazolecarboxaldehyde        derivatives, and benzaldehyde derivatives, thereby forming a        compound wherein

The disclosed compounds have the structure of Formula (I) wherein:

-   -   R₁, R₄, and R₆, are each separately selected from the group        consisting of a hydrogen atom, a halogen atom, and saturated        C₁-C₂₄ alkyl, unsaturated C₁-C₂₄ alkenyl, cycloalkyl,        cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl,        heteroaryl, substituted heteroaryl, amino, substituted amino,        nitro, azido, substituted nitro, phenyl, and substituted phenyl        groups, hydroxy, carboxy, —CO—O—R₇, cyano, alkylthio,        halogenated alkyl including polyhalogenated alkyl, halogenated        carbonyl, and carbonyl —CCO—R₇, wherein R₇ is selected from a        hydrogen atom, a halogen atom, and saturated C₁-C₂₄ alkyl,        unsaturated C₁-C₂₄ alkenyl, cycloalkyl, cycloalkenyl, alkoxy,        cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted        heteroaryl, amino, substituted amino, nitro, azido, substituted        nitro, phenyl, and substituted phenyl groups;    -   R₁′ and R¹″ are independently selected from the group consisting        of a hydrogen atom, a halogen atom, and saturated C₁-C₂₄ alkyl,        unsaturated C₁-C₂₄ alkenyl, cycloalkyl, cycloalkenyl, alkoxy,        cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted        heteroaryl, amino, substituted amino, nitro, azido, substituted        nitro, phenyl, and substituted phenyl groups, hydroxy, carboxy,        —CO—O—R₇, cyano, alkylthio, halogenated alkyl including        polyhalogenated alkyl, halogenated carbonyl, and carbonyl        —CCO—R₇, wherein R₇ is selected from a hydrogen atom, a halogen        atom, and saturated C₁-C₂₄ alkyl, unsaturated C₁-C₂₄ alkenyl,        cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted        aryl, heteroaryl, substituted heteroaryl, amino, substituted        amino, nitro, azido, substituted nitro, phenyl, and substituted        phenyl groups;    -   R₂, R₃, and R₅ are each separately selected from the group        consisting of a hydrogen atom, a halogen atom, and saturated        C₁-C₁₂ alkyl, unsaturated C₁-C₁₂ alkenyl, acyl, cycloalkyl,        alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl,        substituted heteroaryl, amino, substituted amino, nitro, and        substituted nitro groups, sulfonyl and substituted sulfonyl        groups;    -   X₁ and X₂ are separately selected from the group consisting of        an oxygen atom, a nitrogen atom, and a sulfur atom, each either        unsubstituted or substituted with a R₅ group, as defined above;    -   Y is selected from the group consisting of a nitrogen atom, a        substituted nitrogen atom with a R₅ group from above, an oxygen        atom, a sulfur atom, a oxidized sulfur atom, a methylene group        and a substituted methylene group;    -   n is an integer equal to zero, one or two;    -   Z, for each separate n, if non-zero, and Z₁, Z₂, Z₃ and Z₄ are        each separately selected from a carbon atom, a sulfur atom, a        nitrogen atom or an oxygen atom; and    -   the dashed bonds may be either single or double bonds.

In preferred embodiments of the compound and method, theimidazolecarboxaldehyde is5-(1,1-dimethyl-2-ethyl)imidazole-4-carboxaldehyde and the benzaldehydecomprises a single methoxy group. Additional preferred embodiments ofthe compounds described herein include compounds having a t-butyl group,a dimethoxy group, a chloro-group, and a methylthiophen group, andmethods of making such compounds, as well as the compounds described inTables 2, 3 and 4, as well as methods of making such compounds.

Also disclosed are methods and materials for treating neoplastic tissueor preventing cancers or infection by a pathogenic fungus. These methodsand materials are particularly well suited for treatment of mammaliansubjects, more particularly humans, and involve administering to thesubject a dehydrophenylahistin or its analog. The method comprisesadministering to the subject a composition comprising an effectiveantitumor or antifungal amount of a dehydrophenylahistin or its analog.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and form part ofthe specification, merely illustrate certain preferred embodiments ofthe present invention. Together with the remainder of the specification,they are meant to serve to explain preferred modes of making certaincompounds of the invention to those of skilled in the art. In thedrawings:

FIG. 1 illustrates a reaction scheme for producing dehydrophenylahistinsby reacting a diacyldiketopiperazine 1 with an imidazolecarboxaldeheyde2 to yield an intermediate compound 3 which is reacted with abenzaldehyde 4 to produce a dehydrophenylahistin.

FIG. 2 depicts the HPLC profile of the synthetic crudedehydrophenylahistin.

FIG. 3 illustrates a reaction scheme for producing dehydrophenylahistinsby reacting a diacyldiketopiperazine 1 with a benzaldehyde 4 to yield anintermediate compound 17 which is reacted with animidazolecarboxaldeheyde 15 to produce a dehydrophenylahistin.

FIG. 4 depicts the HPLC profiles of the crude synthetictBu-dehyrophenylahistin produced from Route A and from Route B.

FIG. 5 illustrates two modification strategies for dehydroPLH for potentcytotoxic activity.

FIG. 6 depicts the putative active conformation of dehydroPLH at thephenyl moiety.

FIG. 7 depicts Cytochrome P450 metabolism of phenylahistin.

FIG. 8 illustrates the Z-E migration of tBu-dehydroPLH.

FIG. 9 depicts the synthesis and prodrug image of acyl-E-tBu-dehydroPLH.

FIG. 10 depicts the temperature gradient of3-Z-Benzylidene-6-[5″-(1,1-dimethylallyl)-1H-imidazol-4″-Z-ylmethylene]-piperazine-2,5-dione.

FIG. 11 depicts the temperature gradient of3-Z-benzylidene-6-(5″-tert-butyl-1H-imidazol-4″-Z-ylmethylene)-piperazine-2,5-dione.

FIG. 12 depicts the effect of KPU-2, KPU-35 and t-butyl-phenylahistin incomparison to colchicine and taxol on HuVEC monolayer permeability toFITC-Dextran.

FIG. 13 depicts the effect of KPU-2 alone and in combination with CPT-11on estimated tumor growth in the HT-29 Human Colon Tumor Xenograftmodel.

FIG. 14 depicts the effect of KPU-2 alone and in combination with CPT-11on the weight of tumors excised at autopsy in individual mice in theHT-29 Human Colon Tumor Xenograft model.

FIG. 15 depicts the effect of KPU-2 alone and in combination with CPT-11on estimated tumor growth in the HT-29 Human Colon Tumor Xenograftmodel.

FIG. 16 depicts the effect of KPU-2 alone and in combination with CPT-11on the weight of tumors excised at autopsy in individual mice in theHT-29 Human Colon Tumor Xenograft model.

FIG. 17 depicts the effects of: A. KPU-2, B. KPU-35 and C.t-butyl-phenylahistin alone and in combination with CPT-11 on estimatedtumor growth in the HT-29 human colon tumor xenograft model.

FIG. 18 depicts the effects of A. KPU-2, B. KPU-35 and C.t-butyl-phenylahistin alone and in combination with CPT-11 on the weightof tumors excised at autopsy in individual mice in the HT-29 Human ColonTumor Xenograft model.

FIG. 19 depicts the effects of KPU-2 alone and in combination withCPT-11 on tumor growth in the HT-29 human colon tumor xenograft model:comparison of three studies.

FIG. 20 depicts the effects of KPU-2 alone and in combination withCPT-11 on final tumor weights in the HT-29 human colon tumor xenograftmodel: comparison of three studies.

FIG. 21 depicts the effects of KPU-2 alone or in combination withTaxotere on estimated tumor growth in the DU-145 Human Prostate TumorXenograft Model.

FIG. 22 depicts the effects of A. KPU-2, B. KPU-35 and C.t-butyl-phenylahistin alone and in combination with Taxotere on theestimated tumor growth based on observations made during the in-lifeportion of the DU-145 Human Prostate Tumor Xenograft Model.

FIG. 23 depicts the effects of KPU-2 alone and in combination withTaxotere on the individual excised tumor weights at autopsy in theDU-145 Human Prostate Tumor Xenograft Model.

FIG. 24 depicts the effects of KPU-35 alone and in combination withTaxotere on the individual excised tumor weights at autopsy in theDU-145 Human Prostate Tumor Xenograft Model.

FIG. 25 depicts the effects of A. KPU-2, B. KPU-35 and C.t-butyl-phenylahistin alone and in combination with Taxotere in MCF-7Human Breast Tumor Xenograft model.

FIG. 26 depicts the effects of KPU-2 alone and in combination withTaxotere on estimated tumor growth in the A549 Human Lung TumorXenograft model.

FIG. 27 depicts the effects of KPU-2 alone and in combination withTaxotere on the excised tumor weights at autopsy in the A549 Human LungTumor Xenograft model.

FIG. 28 depicts the effects of KPU-2 alone and in combination withPaclitaxel on estimated tumor weight in the murine mammary fat padimplanted MDA-231 Human Breast Tumor model.

FIG. 29 depicts effects of A. KPU-2, B. KPU-35 and C.t-butyl-phenylahistin alone and in combination with Paclitaxel in theMurine Melanoma B16 F10 Metastatic Tumor Model. In certain Figures,compounds are identified using an alternative designation. A completechart to convert these alternative designations is as follows:Alternative Designation designation used herein NPI-2350(−)-phenylahistin NPI-2352 KPU-01 NPI-2353 KPU-03 NPI-2354 KPU-04NPI-2355 KPU-05 NPI-2356 KPU-06 NPI-2357 KPU-07 NPI-2358 KPU-02 NPI-2359KPU-08 NPI-2360 KPU-09 NPI-2361 KPU-10 NPI-2362 KPU-11 NPI-2363 KPU-12NPI-2364 KPU-13 NPI-2365 KPU-14 NPI-2366 KPU-15 NPI-2367 KPU-16 NPI-2368KPU-17 NPI-2369 KPU-18 NPI-2370 KPU-19 NPI-2371 KPU-21 NPI-2372 KPU-22NPI-2373 KPU-23 NPI-2374 KPU-24 NPI-2375 KPU-25 NPI-2376 KPU-28 NPI-2377KPU-26 NPI-2378 KPU-27 NPI-2379 KPU-29 NPI-2380 KPU-20 NPI-2381 KPU-30NPI-2382 KPU-31 NPI-2383 KPU-32 NPI-2384 KPU-33 NPI-2385 KPU-34 NPI-2386KPU-35 NPI-2387 KPU-36 NPI-2388 KPU-37 NPI-2389 KPU-38 NPI-2390 KPU-39NPI-2391 KPU-40 NPI-2392 KPU-41 NPI-2393 KPU-42 NPI-2394 KPU-43 NPI-2395KPU-44 NPI-2396 KPU-45 NPI-2397 KPU-46 NPI-2398 KPU-47 NPI-2399 KPU-48NPI-2400 KPU-49 NPI-2401 KPU-50 NPI-2402 KPU-51 NPI-2403 KPU-52 NPI-2404KPU-53 NPI-2405 KPU-54 NPI-2406 KPU-55 NPI-2407 KPU-56 NPI-2408 KPU-57NPI-2409 KPU-58 NPI-2410 KPU-59 NPI-2411 KPU-60 NPI-2412 KPU-61 NPI-2413KPU-62 NPI-2414 KPU-63 NPI-2415 KPU-64 NPI-2416 KPU-65 NPI-2417 KPU-66NPI-2418 KPU-67 NPI-2419 KPU-68 NPI-2420 KPU-69 NPI-2421 KPU-70 NPI-2422KPU-71 NPI-2423 KPU-72 NPI-2424 KPU-73 NPI-2425 KPU-74 NPI-2426 KPU-75NPI-2427 KPU-76 NPI-2428 KPU-77 NPI-2429 KPU-79 NPI-2430 KPU-80 NPI-2431KPU-81 NPI-2432 KPU-82 NPI-2433 KPU-83 NPI-2434 KPU-84 NPI-2435 KPU-86NPI-2436 KPU-87 NPI-2437 KPU-88 NPI-2438 KPU-89 NPI-2439 KPU-90 NPI-2440KPU-91 NPI-2441 KPU-92 NPI-2442 KPU-80 NPI-2455 KPU-94 NPI-2456 KPU-95NPI-2457 KPU-96 NPI-2458 KPU-97 NPI-2459 KPU-98 NPI-2460 t-butylphenylahistin

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Each reference cited herein, including the U.S. patents cited herein, isto be considered incorporated by reference in its entirety into thisspecification, to the full extent permissible by law.

The disclosure provides methods for the synthetic preparation ofcompounds, including novel compounds, including dehydrophenyalahistinand dehydrophenyalahistin analogs, and provides methods for producingpharmaceutically acceptable cell cycle inhibitors, antitumor agents andantifungal agents in relatively high yield, wherein said compoundsand/or their derivatives are among the active ingredients in these cellcycle inhibitors, antitumor agents and antifungal agents. Other objectsinclude providing novel compounds not obtainable by currently available,non-synthetic methods. It is also an object to provide a method oftreating cancer, particularly human cancer, comprising the step ofadministering an effective tumor-growth inhibiting amount of a member ofa class of new anti-tumor compounds. This invention also provides amethod for preventing or treating a pathogenic fungus in a subject whichinvolves administering to the subject an effective anti-fungal amount ofa member of a class of new anti-fungal compounds, e.g., administering adehydrophenylahistin or its analog in an amount and manner whichprovides the intended antifungal effect. In the preferred embodiment ofthe compounds and methods of making and using such compounds disclosedherein, but not necessarily in all embodiments of the present invention,these objectives are met.

Disclosed herein, also, are compounds, and methods of producing a classof compounds, wherein the compounds are represented by Formula (I):

wherein:

-   -   R₁, R₄, and R₆, are each separately selected from the group        consisting of a hydrogen atom, a halogen atom, and saturated        C₁-C₂₄ alkyl, unsaturated C₁-C₂₄ alkenyl, cycloalkyl,        cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl,        heteroaryl, substituted heteroaryl, amino, substituted amino,        nitro, azido, substituted nitro, phenyl, and substituted phenyl        groups, hydroxy, carboxy, —CO—O—R₇, cyano, alkylthio,        halogenated alkyl including polyhalogenated alkyl, halogenated        carbonyl, and carbonyl —CCO—R₇, wherein R₇ is selected from a        hydrogen atom, a halogen atom, and saturated C₁-C₂₄ alkyl,        unsaturated C₁-C₂₄ alkenyl, cycloalkyl, cycloalkenyl, alkoxy,        cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted        heteroaryl, amino, substituted amino, nitro, azido, substituted        nitro, phenyl, and substituted phenyl groups;    -   R₁′ and R¹″ are independently selected from the group consisting        of a hydrogen atom, a halogen atom, and saturated C₁-C₂₄ alkyl,        unsaturated C₁-C₂₄ alkenyl, cycloalkyl, cycloalkenyl, alkoxy,        cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted        heteroaryl, amino, substituted amino, nitro, azido, substituted        nitro, phenyl, and substituted phenyl groups, hydroxy, carboxy,        —CO—O—R₇, cyano, alkylthio, halogenated alkyl including        polyhalogenated alkyl, halogenated carbonyl, and carbonyl        —CCO—R₇, wherein R₇ is selected from a hydrogen atom, a halogen        atom, and saturated C₁-C₂₄ alkyl, unsaturated C₁-C₂₄ alkenyl,        cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted        aryl, heteroaryl, substituted heteroaryl, amino, substituted        amino, nitro, azido, substituted nitro, phenyl, and substituted        phenyl groups;    -   R, R₁′ and R₁″ are either covalently bound to one another or are        not covalently bound to one another;    -   R₂, R₃, and R₅ are each separately selected from the group        consisting of a hydrogen atom, a halogen atom, and saturated        C₁-C₁₂ alkyl, unsaturated C₁-C₁₂ alkenyl, acyl, cycloalkyl,        alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl,        substituted heteroaryl, amino, substituted amino, nitro, and        substituted nitro groups, sulfonyl and substituted sulfonyl        groups;    -   X₁ and X₂ are separately selected from the group consisting of        an oxygen atom, a nitrogen atom, and a sulfur atom, each either        unsubstituted or substituted with a R₅ group, as defined above;    -   Y is selected from the group consisting of a nitrogen atom, a        substituted nitrogen atom with a R₅ group from above, an oxygen        atom, a sulfur atom, a oxidized sulfur atom, a methylene group        and a substituted methylene group;    -   n is an integer equal to zero, one or two;    -   Z, for each separate n, if non-zero, and Z₁, Z₂, Z₃ and Z₄ are        each separately selected from a carbon atom, a sulfur atom, a        nitrogen atom or an oxygen atom; and    -   the dashed bonds may be either single or double bonds.

The method comprises a method of producing compounds of Formula (I) bythe steps of:

-   -   reacting a diacyldiketopiperazine with a first aldehyde to        produce an intermediate compound; and    -   reacting said intermediate compound with a second aldehyde to        produce said class of compounds with said generic structure,        wherein    -   said first aldehyde and said second aldehydes are selected from        the group consisting of an oxazolecarboxaldeyhyde,        imidazolecarboxaldehyde, a benzaldehyde, imidazolecarboxaldehyde        derivatives, and benzaldehyde derivatives, thereby forming a        compound of Formula (I) wherein    -   R₁, R₄, and R₆, are each separately selected from the group        consisting of a hydrogen atom, a halogen atom, and saturated        C₁-C₂₄ alkyl, unsaturated C₁-C₂₄ alkenyl, cycloalkyl,        cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl,        heteroaryl, substituted heteroaryl, amino, substituted amino,        nitro, azido, substituted nitro, phenyl, and substituted phenyl        groups, hydroxy, carboxy, —CO—O—R₇, cyano, alkylthio,        halogenated alkyl including polyhalogenated alkyl, halogenated        carbonyl, and carbonyl —CCO—R₇, wherein R₇ is selected from a        hydrogen atom, a halogen atom, and saturated C₁-C₂₄ alkyl,        unsaturated C₁-C₂₄ alkenyl, cycloalkyl, cycloalkenyl, alkoxy,        cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted        heteroaryl, amino, substituted amino, nitro, azido, substituted        nitro, phenyl, and substituted phenyl groups;    -   R₁′ and R₁″ are independently is selected from the group        consisting of a hydrogen atom, a halogen atom, and saturated        C₁-C₂₄ alkyl, unsaturated C₁-C₂₄ alkenyl, cycloalkyl,        cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl,        heteroaryl, substituted heteroaryl, amino, substituted amino,        nitro, azido, substituted nitro, phenyl, and substituted phenyl        groups, hydroxy, carboxy, —CO—O—R₇, cyano, alkylthio,        halogenated alkyl including polyhalogenated alkyl, halogenated        carbonyl, and carbonyl —CCO—R₇, wherein R₇ is selected from a        hydrogen atom, a halogen atom, and saturated C₁-C₂₄ alkyl,        unsaturated C₁-C₂₄ alkenyl, cycloalkyl, cycloalkenyl, alkoxy,        cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted        heteroaryl, amino, substituted amino, nitro, azido, substituted        nitro, phenyl, and substituted phenyl groups;    -   R₂, R₃, and R₅ are each separately selected from the group        consisting of a hydrogen atom, a halogen atom, and saturated        C₁-C₁₂ alkyl, unsaturated C₁-C₁₂ alkenyl, acyl, cycloalkyl,        alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl,        substituted heteroaryl, amino, substituted amino, nitro, and        substituted nitro groups, sulfonyl and substituted sulfonyl        groups;    -   X₁ and X₂ are separately selected from the group consisting of        an oxygen atom, a nitrogen atom and a sulfur atom, and    -   Y is selected from the group consisting of a nitrogen atom, a        substituted nitrogen atom with a R₅ group from above, an oxygen        atom, a sulfur atom, a oxidized sulfur atom, a methylene group        and a substituted methylene group;    -   Z, for each separate n, if non-zero, and Z₁, Z₂, Z₃ and Z₄ are        each separately selected from a carbon atom, a sulfur atom, a        nitrogen atom or an oxygen atom; and    -   the dashed bonds may be either single or double bonds.

Also provided are pharmaceutically acceptable salts and pro-drug estersof the compound of Formulae (I) and (II) and provides methods ofsynthesizing such compounds by the methods disclosed herein.

The term “pro-drug ester,” especially when referring to a pro-drug esterof the compound of Formula (I) synthesized by the methods disclosedherein, refers to a chemical derivative of the compound that is rapidlytransformed in vivo to yield the compound, for example, by hydrolysis inblood or inside tissues. The term “pro-drug ester” refers to derivativesof the compounds disclosed herein formed by the addition of any ofseveral ester-forming groups that are hydrolyzed under physiologicalconditions. Examples of pro-drug ester groups include pivoyloxymethyl,acetoxymethyl, phthalidyl, indanyl and methoxymethyl, as well as othersuch groups known in the art, including a(5-R-2-oxo-1,3-dioxolen-4-yl)methyl group. Other examples of pro-drugester groups can be found in, for example, T. Higuchi and V. Stella, in“Pro-drugs as Novel Delivery Systems”, Vol. 14, A.C.S. Symposium Series,American Chemical Society (1975); and “Bioreversible Carriers in DrugDesign: Theory and Application”, edited by E. B. Roche, Pergamon Press:New York, 14-21 (1987) (providing examples of esters useful as prodrugsfor compounds containing carboxyl groups).

The term “pro-drug ester,” as used herein, also refers to a chemicalderivative of the compound that is rapidly transformed in vivo to yieldthe compound, for example, by hydrolysis in blood. The term “pro-drugester” refers to derivatives of the compounds disclosed herein formed bythe addition of any of several ester-forming groups that are hydrolyzedunder physiological conditions. Examples of pro-drug ester groupsinclude pivoyloxymethyl, acetoxymethyl, phthalidyl, indanyl andmethoxymethyl, as well as other such groups known in the art, includinga (5-R-2-oxo-1,3-dioxolen-4-yl)methyl group. Other examples of pro-drugester groups can be found in, for example, T. Higuchi and V. Stella, in“Pro-drugs as Novel Delivery Systems”, Vol. 14, A.C.S. Symposium Series,American Chemical Society (1975); and “Bioreversible Carriers in DrugDesign: Theory and Application”, edited by E. B. Roche, Pergamon Press:New York, 14-21 (1987) (providing examples of esters useful as prodrugsfor compounds containing carboxyl groups).

The term “pharmaceutically acceptable salt,” especially when referringto a pharmaceutically acceptable salt of the compound of Formula (I)synthesized by the methods disclosed herein, refers to anypharmaceutically acceptable salts of a compound, and preferably refersto an acid addition salt of a compound. Preferred examples ofpharmaceutically acceptable salt are the alkali metal salts (sodium orpotassium), the alkaline earth metal salts (calcium or magnesium), orammonium salts derived from ammonia or from pharmaceutically acceptableorganic amines, for example C₁-C₇ alkylamine, cyclohexylamine,triethanolamine, ethylenediamine or tris-(hydroxymethyl)-aminomethane.With respect to compounds synthesized by the method that are basicamines, the preferred examples of pharmaceutically acceptable salts areacid addition salts of pharmaceutically acceptable inorganic or organicacids, for example, hydrohalic, sulfuric, phosphoric acid or aliphaticor aromatic carboxylic or sulfonic acid, for example acetic, succinic,lactic, malic, tartaric, citric, ascorbic, nicotinic, methanesulfonic,p-toluensulfonic or naphthalenesulfonic acid.

The term “pharmaceutically acceptable salt,” as used herein, also refersto any pharmaceutically acceptable salts of a compound, and preferablyrefers to an acid addition salt of a compound. Preferred examples ofpharmaceutically acceptable salt are the alkali metal salts (sodium orpotassium), the alkaline earth metal salts (calcium or magnesium), orammonium salts derived from ammonia or from pharmaceutically acceptableorganic amines, for example C₁-C₇ alkylamine, cyclohexylamine,triethanolamine, ethylenediamine or tris-(hydroxymethyl)-aminomethane.With respect to compounds that are basic amines, the preferred examplesof pharmaceutically acceptable salts are acid addition salts ofpharmaceutically acceptable inorganic or organic acids, for example,hydrohalic, sulfuric, phosphoric acid or aliphatic or aromaticcarboxylic or sulfonic acid, for example acetic, succinic, lactic,malic, tartaric, citric, ascorbic, nicotinic, methanesulfonic,p-toluensulfonic or naphthalenesulfonic acid.

Preferred pharmaceutical compositions disclosed herein includepharmaceutically acceptable salts and pro-drug esters of the compound ofFormula (I) synthesized by the method disclosed herein. Accordingly, ifthe manufacture of pharmaceutical formulations involves intimate mixingof the pharmaceutical excipients and the active ingredient in its saltform, then it is preferred to use pharmaceutical excipients which arenon-basic, that is, either acidic or neutral excipients.

In preferred embodiments of the methods of the compounds disclosedherein, a relatively rigid, planar pseudo three-ring structure may beformed. To stabilize such a relatively rigid, planar pseudo three-ringstructure, R₃ may preferably chosen to be hydrogen.

In other preferable embodiments of the compounds and methods describedherein, n is equal to zero or one, more preferable one, and Z₂, Z₃, andZ₄, and each separately selected from an oxygen atom, a nitrogen atom,and a carbon atom, more preferable at one least one of Z₂, Z₃, and Z₄being a carbon atom, and most preferable at least two of Z₂, Z₃, and Z₄being a carbon atom. All Z's may simultaneous be carbon atoms.

Still other preferred embodiments of the methods and compositionsdisclosed herein involve compounds having the structures of Formulae(Ia) and (Ib), below:

wherein the variable groups are as defined herein.

The term “halogen atom,” as used herein, means any one of theradio-stable atoms of column 7 of the Periodic Table of the Elements,i.e., fluorine, chlorine, bromine, or iodine, with fluorine and chlorinebeing preferred.

The term “alkyl,” as used herein, means any unbranched or branched,substituted or unsubstituted, saturated hydrocarbon, with C₁-C₆unbranched, saturated, unsubstituted hydrocarbons being preferred, withmethyl, ethyl, iosbutyl, and tert-butyl being most preferred. Among thesubstituted, saturated hydrocarbons, C₁-C₆ mono- and di- and per-halogensubstituted saturated hydrocarbons and amino-substituted hydrocarbonsare preferred, with perfluromethyl, perchloromethyl,perfluoro-tert-butyl, and perchloro-tert-butyl being the most preferred.The term “substituted” has its ordinary meaning, as found in numerouscontemporary patents from the related art. See, for example, U.S. Pat.Nos. 6,583,143, 6,509,331; 6,506,787; 6,500,825; 5,922,683; 5,886,210;5,874,443; and 6,350,759. Specifically, the definition of substituted isas broad as that provided in U.S. Pat. No. 6,583,143, which defines theterm substituted as any groups such as alkyl, aryl, arylalkyl,heteroaryl, heteroarylalkyl, heterocycle and heterocyclealkyl, whereinat least one hydrogen atom is replaced with a substituent. The term“substituted” is also as broad as the definition provided in U.S. Pat.No. 6,509,331, which defines the term “substituted alkyl” such that itrefers to an alkyl group, preferably of from 1 to 10 carbon atoms,having from 1 to 5 substituents, and preferably 1 to 3 substituents,selected from the group consisting of alkoxy, substituted alkoxy,cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino,aminoacyl, aminoacyloxy, oxyacylamino, cyano, halogen, hydroxyl,carboxyl, carboxylalkyl, keto, thioketo, thiol, thioalkoxy, substitutedthioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic,heterocyclooxy, hydroxyamino, alkoxyamino, nitro, —SO-alkyl,—SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl. The otherabove-listed patents also provide standard definitions for the term“substituted” that are well-understood by those of skill in the art. Theterm “cycloalkyl” refers to any non-aromatic hydrocarbon ring,preferably having five to twelve atoms comprising the ring. The term“acyl” refers to alkyl or aryl groups derived from an oxoacid, with anacetyl group being preferred.

The term “alkenyl,” as used herein, means any unbranched or branched,substituted or unsubstituted, unsaturated hydrocarbon includingpolyunsaturated hydrocarbons, with C₁-C₆ unbranched, mono-unsaturatedand di-unsaturated, unsubstituted hydrocarbons being preferred, andmono-unsaturated, di-halogen substituted hydrocarbons being mostpreferred. In the R₁ and R₄ positions, of the compound of structure (I)a z-isoprenyl moiety is particularly preferred. The term “cycloalkenyl”refers to any non-aromatic hydrocarbon ring, preferably having five totwelve atoms comprising the ring.

The terms “aryl,” “substituted aryl,” “heteroaryl,” and “substitutedheteroaryl,” as used herein, refer to aromatic hydrocarbon rings,preferably having five, six, or seven atoms, and most preferably havingsix atoms comprising the ring. “Heteroaryl” and “substitutedheteroaryl,” refer to aromatic hydrocarbon rings in which at least oneheteroatom, e.g., oxygen, sulfur, or nitrogen atom, is in the ring alongwith at least one carbon atom.

The term “alkoxy” refers to any unbranched, or branched, substituted orunsubstituted, saturated or unsaturated ether, with C₁-C₆ unbranched,saturated, unsubstituted ethers being preferred, with methoxy beingpreferred, and also with dimethyl, diethyl, methyl-isobutyl, andmethyl-tert-butyl ethers also being preferred. The term “cycloalkoxy”refers to any non-aromatic hydrocarbon ring, preferably having five totwelve atoms comprising the ring.

The terms “purified,” “substantially purified,” and “isolated” as usedherein refer to the compound being free of other, dissimilar compoundswith which the compound is normally associated in its natural state, sothat the compound of the invention comprises at least 0.5%, 1%, 5%, 10%,or 20%, and most preferably at least 50% or 75% of the mass, by weight,of a given sample.

The compound of Formula (I) may be chemically synthesized or producedfrom reagents known and available in the art. For example, modificationsof diacyldiketopiperazine (diacetyldiketopiperazine) have beendescribed, for example, by Loughlin et al., 2000 Bioorg Med Chem Lett10:91 or by Brocchini et al. in WO 95/21832. The diacyldiketopiperazine(diacetyldiketopiperazine) may be prepared, for example, bydiacetylation of inexpensive 2,5-piperazinedione (TCI Cat. No. GO100, 25g) with sodium acetate and sodium anhydride. The diacetyl structure ofthe activated deketopiperazine can be replaced with other acyl groups,to include carbamates such as Boc (t-butoxycarbonyl), Z(benzoyloxycarbonyl).

The imidazolecarboxaldehyde may be prepared, for example, according theprocedure disclosed in Hayashi et al., 2000 J Organic Chem 65: 8402 asdepicted below:

Another example of an imidazolecarboxaldehyde derivative is animidazole-4-carboxaldehyde 15 derivative which can be produced from, forexample, a commercially available beta-ketoester 18 (TCI Cat, No. P1031,25 mL) by the following route:

The synthetic method disclosed herein may be preferably performed in thepresence of cesium carbonate as a base in DMF and in a deoxygenatedatmosphere. The inert atmosphere circumvents the probable oxidation ofactivated α-carbon atoms of the diketopiperazine ring during thetreatment with cesium carbonate (see below) as reported, for example, byWatanabe et al., 18^(th) International Congress of HeterocyclicChemistry in Yokohama, Japan (Jul. 30, 2001), Abstract, page 225.

Air-Oxidation of Activated Carbonyl Compounds with Cesium Salts

Other embodiments of the synthetic method involve modifications to thecompounds used in or otherwise involved in the synthesis of compoundsrepresented by Formula (I). Such derivatives may include modificationsto the phenyl ring, introduction of other aromatic ring systems,position of the aromatic ring, alterations to the imidazole ring systemand/or further modifications to the 5-position on the imidazole ring.Examples of such modifications are discussed, for example, in Example 4.The result of such modifications include increased nitrogen content ofthe phenyl ring and/or the compound which may increase compoundsolubility. Other modifications may incorporate derivatives of knowntubulin inhibitors, thereby mimicking the activity of the tubulininhibitors. Other modifications may simplify the synthesis of theβ-ketoester involved in the production of the imidazolecarboxaldehydeused in the methods disclosed herein.

Pharmaceutical Compositions

The present invention also encompasses the compounds disclosed herein,optionally and preferably produced by the methods disclosed herein, inpharmaceutical compositions comprising a pharmaceutically acceptablecarrier prepared for storage and subsequent administration, which have apharmaceutically effective amount of the products disclosed above in apharmaceutically acceptable carrier or diluent. Acceptable carriers ordiluents for therapeutic use are well known in the pharmaceutical art,and are described, for example, in Remington's Pharmaceutical Sciences,Mack Publishing Co. (A. R. Gennaro edit. 1985). Preservatives,stabilizers, dyes and even flavoring agents may be provided in thepharmaceutical composition. For example, sodium benzoate, ascorbic acidand esters of p-hydroxybenzoic acid may be added as preservatives. Inaddition, antioxidants and suspending agents may be used.

The dehydrophenylahistin or dehydrophenylahistin analog compositions maybe formulated and used as tablets, capsules, or elixirs for oraladministration; suppositories for rectal administration; sterilesolutions, suspensions for injectable administration; patches fortransdermal administration, and sub-dermal deposits and the like.Injectables can be prepared in conventional forms, either as liquidsolutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection or infusion, or as emulsions.Suitable excipients are, for example, water, saline, dextrose, mannitol,lactose, lecithin, albumin, sodium glutamate, cysteine hydrochloride,human serum albumin and the like. In addition, if desired, theinjectable pharmaceutical compositions may contain minor amounts ofnontoxic auxiliary substances, such as wetting agents, pH bufferingagents, and the like. If desired, absorption enhancing preparations (forexample, liposomes), may be utilized.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or other organic oilssuch as soybean, grapefruit or almond oils, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents that increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Pharmaceutical preparations for oral use may be obtained by combiningthe active compounds with solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. Dragee cores areprovided with suitable coatings. For this purpose, concentrated sugarsolutions may be used, which may optionally contain gum arabic, talc,polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for identification or to characterize differentcombinations of active compound doses. For this purpose, concentratedsugar solutions may be used, which may optionally contain gum arabic,talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for identification or to characterize differentcombinations of active compound doses. Such formulations can be madeusing methods known in the art (see, for example, U.S. Pat. No.5,733,888 (injectable compositions); U.S. Pat. No. 5,726,181 (poorlywater soluble compounds); U.S. Pat. No. 5,707,641 (therapeuticallyactive proteins or peptides); U.S. Pat. No. 5,667,809 (lipophilicagents); U.S. Pat. No. 5,576,012 (solubilizing polymeric agents); U.S.Pat. No. 5,707,615 (anti-viral formulations); U.S. Pat. No. 5,683,676(particulate medicaments); U.S. Pat. No. 5,654,286 (topicalformulations); U.S. Pat. No. 5,688,529 (oral suspensions); U.S. Pat. No.5,445,829 (extended release formulations); U.S. Pat. No. 5,653,987(liquid formulations); U.S. Pat. No. 5,641,515 (controlled releaseformulations) and U.S. Pat. No. 5,601,845 (spheroid formulations).

Further disclosed herein are various pharmaceutical compositions wellknown in the pharmaceutical art for uses that include intraocular,intranasal, and intraauricular delivery. Pharmaceutical formulationsinclude aqueous ophthalmic solutions of the active compounds inwater-soluble form, such as eyedrops, or in gellan gum (Shedden et al.,2001 Clin Ther 23(3):440-50) or hydrogels (Mayer et al., 1996Ophthalmologica 210:101-3); ophthalmic ointments; ophthalmicsuspensions, such as microparticulates, drug-containing small polymericparticles that are suspended in a liquid carrier medium (Joshi, A., 1994J Ocul Pharmacol 10:29-45), lipid-soluble formulations (Alm et al., 1989Prog Clin Biol Res 312:447-58), and microspheres (Mordenti, 1999 ToxicolSci 52:101-6); and ocular inserts. Such suitable pharmaceuticalformulations are most often and preferably formulated to be sterile,isotonic and buffered for stability and comfort. Pharmaceuticalcompositions may also include drops and sprays often prepared tosimulate in many respects nasal secretions to ensure maintenance ofnormal ciliary action. As disclosed in Remington's PharmaceuticalSciences (Mack Publishing, 18^(th) Edition), and well-known to thoseskilled in the art, suitable formulations are most often and preferablyisotonic, slightly buffered to maintain a pH of 5.5 to 6.5, and mostoften and preferably include antimicrobial preservatives and appropriatedrug stabilizers. Pharmaceutical formulations for intraauriculardelivery include suspensions and ointments for topical application inthe ear. Comnnon solvents for such aural formulations include glycerinand water.

When used as a cell cycle inhibitor, a tumor-growth-inhibiting, or afungus-growth-inhibiting compound, the compound of Formula (I) can beadministered by either oral or a non-oral pathways. When administeredorally, it can be administered in capsule, tablet, granule, spray,syrup, or other such form. When administered non-orally, it can beadministered as an aqueous suspension, an oily preparation or the likeor as a drip, suppository, salve, ointment or the like, whenadministered via injection or infusion, subcutaneously,intreperitoneally, intravenously, intramuscularly, or the like.Similarly, it may be administered topically, rectally, or vaginally, asdeemed appropriate by those of skill in the art for bringing thecompound into optimal contact with a tumor, thus inhibiting the growthof the tumor. Local administration at the site of the tumor is alsocontemplated, either before or after tumor resection, as are controlledrelease formulations, depot formulations, and infusion pump delivery.

Methods of Administration

The present invention also encompasses methods for making and foradministering the disclosed chemical compounds and the disclosedpharmaceutical compositions. Such disclosed methods include, amongothers, (a) administration though oral pathways, which administrationincludes administration in capsule, tablet, granule, spray, syrup, orother such forms; (b) administration through non-oral pathways, whichadministration includes administration as an aqueous suspension, an oilypreparation or the like or as a drip, suppository, salve, ointment orthe like; administration via injection or infusion, subcutaneously,intraperitoneally, intravenously, intramuscularly, intradermally, or thelike; as well as (c) administration topically, (d) administrationrectally, or (e) administration vaginally, as deemed appropriate bythose of skill in the art for bringing the compound into contact withliving tissue; and (f) administration via controlled releasedformulations, depot formulations, and infusion pump delivery. As furtherexamples of such modes of administration and as further disclosure ofmodes of administration, disclosed herein are various methods foradministration of the disclosed chemical compounds and pharmaceuticalcompositions including modes of administration through intraocular,intranasal, and intraauricular pathways.

The pharmaceutically effective amount of the dehydrophenylahistin ordehydrophenylahistin analog composition required as a dose will dependon the route of administration, the type of animal, including human,being treated, and the physical characteristics of the specific animalunder consideration. The dose can be tailored to achieve a desiredeffect, but will depend on such factors as weight, diet, concurrentmedication and other factors which those skilled in the medical artswill recognize.

In practicing the methods, the products or compositions can be usedalone or in combination with one another, or in combination with othertherapeutic or diagnostic agents. For example, as disclosed herein, thecompounds disclosed herein are effective in the treatment of cancer whenused in combination with other actives, specifically otherchemotherapeutics, for example biologics and the specificchemotherapeutics CPT-11, Taxotene (docataxel) and pacitaxel. Thecompounds disclosed herein are also effective in the treatment of cancerwhen used in combination with other actives, including anti-vascularagents, anti-angiogenenic agents, such as Erbuitux(Imclone/bristol-Myers) and Iressa (AstraZeneca), other VEGF inhibitorsand biologics, more specifically, at least one anti-VEGF antibodies,especially monoclonal antibodies to the VEGF receptor, including DC101,a rat monoclonal antibody, which blocks the mouse VEGF receptor 2(flk-1). Such combinations may be utilized in vivo, ordinarily in amammal, preferably in a human, or in vitro. In employing them in vivo,the disclosed compounds, alone or in combination with otherchemotherapeutics or other biologic products, may be administered to themammal in a variety of ways, including parenterally, intravenously, viainfusion or injection, subcutaneously, intramuscularly, colonically,rectally, vaginally, nasally or intraperitoneally, employing a varietyof dosage forms. Such methods may also be applied to testing chemicalactivity in vivo.

As will be readily apparent to one skilled in the art, the useful invivo dosage to be administered and the particular mode of administrationwill vary depending upon the age, weight and mammalian species treated,the particular compounds employed, and the specific use for which thesecompounds are employed. The determination of effective dosage levels,that is the dosage levels necessary to achieve the desired result, canbe accomplished by one skilled in the art using routine pharmacologicalmethods. Typically, human clinical applications of products arecommenced at lower dosage levels, with dosage level being increaseduntil the desired effect is achieved. Alternatively, acceptable in vitrostudies can be used to establish useful doses and routes ofadministration of the compositions identified by the present methodsusing established pharmacological methods.

In non-human animal studies, applications of potential products arecommenced at higher dosage levels, with dosage being decreased until thedesired effect is no longer achieved or adverse side effects disappear.The dosage may range broadly, depending upon the desired affects and thetherapeutic indication. Typically, dosages may be between about 10microgram/kg and 100 mg/kg body weight, preferably between about 100microgram/kg and 10 mg/kg body weight. Alternatively dosages may bebased and calculated upon the surface area of the patient, as understoodby those of skill in the art. Administration may be oral on an everythird da6y, aevery other day, daily, twice daily, or thirce daily basis.

The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition. See forexample, Fingl et al., in The Pharmacological Basis of Therapeutics,1975. It should be noted that the attending physician would know how toand when to terminate, interrupt, or adjust administration due totoxicity, or to organ dysfunctions. Conversely, the attending physicianwould also know to adjust treatment to higher levels if the clinicalresponse were not adequate (precluding toxicity). The magnitude of anadministrated dose in the management of the disorder of interest willvary with the severity of the condition to be treated and to the routeof administration. The severity of the condition may, for example, beevaluated, in part, by standard prognostic evaluation methods. Further,the dose and perhaps dose frequency, will also vary according to theage, body weight, and response of the individual patient. A programcomparable to that discussed above may be used in veterinary medicine.

Depending on the specific conditions being treated, such agents may beformulated and administered systemically or locally. A variety oftechniques for formulation and administration may be found inRemington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co.,Easton, Pa. (1990). Suitable administration routes may include oral,rectal, transdermal, vaginal, transmucosal, or intestinaladministration; parenteral delivery, including intramuscular,subcutaneous, intramedullary injections, as well as intrathecal, directintraventricular, intravenous, via infusion, intraperitoneal,intranasal, or intraocular injections.

For injection or infusion, the agents may be formulated in aqueoussolutions, for example, in physiologically compatible buffers such asHanks' solution, Ringer's solution, or physiological saline buffer. Forsuch transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art. Use of pharmaceutically acceptable carriersto formulate the compounds herein disclosed for the practice of theinvention into dosages suitable for systemic administration is withinthe scope of the invention. With proper choice of carrier and suitablemanufacturing practice, the compositions disclosed herein, inparticular, those formulated as solutions, may be administeredparenterally, such as by intravenous injection or infusion. Thecompounds can be formulated readily using pharmaceutically acceptablecarriers well known in the art into dosages suitable for oraladministration. Such carriers enable the compounds to be formulated astablets, pills, capsules, liquids, gels, syrups, slurries, suspensionsand the like, for oral ingestion by a patient to be treated.

Agents intended to be administered intracellularly may be administeredusing techniques well known to those of ordinary skill in the art. Forexample, such agents may be encapsulated into liposomes, thenadministered as described above. All molecules present in an aqueoussolution at the time of liposome formation are incorporated into theaqueous interior. The liposomal contents are both protected from theexternal micro-environment and, because liposomes fuse with cellmembranes, are efficiently delivered into the cell cytoplasm.Additionally, due to their hydrophobicity, small organic molecules maybe directly administered intracellularly.

Determination of the effective amounts is well within the capability ofthose skilled in the art, especially in light of the detailed disclosureprovided herein. In addition to the active ingredients, thesepharmaceutical compositions may contain suitable pharmaceuticallyacceptable carriers comprising excipients and auxiliaries whichfacilitate processing of the active compounds into preparations whichcan be used pharmaceutically. The preparations formulated for oraladministration may be in the form of tablets, dragees, capsules, orsolutions. The pharmaceutical compositions may be manufactured in amanner that is itself known, for example, by means of conventionalmixing, dissolving, granulating, dragee-making, levitating, emulsifying,encapsulating, entrapping, or lyophilizing processes.

Compounds disclosed herein can be evaluated for efficacy and toxicityusing known methods. For example, the toxicology of a particularcompound, or of a subset of the compounds, sharing certain chemicalmoieties, may be established by determining in vitro toxicity towards acell line, such as a mammalian, and preferably human, cell line. Theresults of such studies are often predictive of toxicity in animals,such as mammals, or more specifically, humans. Alternatively, thetoxicity of particular compounds in an animal model, such as mice, rats,rabbits, or monkeys, may be determined using known methods. The efficacyof a particular compound may be established using several art recognizedmethods, such as in vitro methods, animal models, or human clinicaltrials. Art-recognized in vitro models exist for nearly every class ofcondition, including the conditions abated by the compounds disclosedherein, including cancer, cardiovascular diseasae and various fungalinfections. Similarly, acceptable animal models may be used to establishefficacy of chemicals to treat such conditions. When selecting a modelto determine efficacy, the skilled artisan can be guided by the state ofthe art to choose an appropriate model, dose, and route ofadministration, and regime. Of course, human clinical trials can also beused to determine the efficacy of a compound in humans.

When used as an anti-cancer agent, or a tumor-growth-inhibitingcompound, the compounds disclosed herein may be administered by eitheroral or a non-oral pathways. When administered orally, it can beadministered in capsule, tablet, granule, spray, syrup, or other suchform. When administered non-orally, it can be administered as an aqueoussuspension, an oily preparation or the like or as a drip, suppository,salve, ointment or the like, when administered via injection orinfusion, subcutaneously, intreperitoneally, intravenously,intramuscularly, intradermally, or the like. Similarly, it may beadministered topically, rectally, or vaginally, as deemed appropriate bythose of skill in the art for bringing the compound into optimal contactwith a tumor, thus inhibiting the growth of the tumor. Localadministration at the site of the tumor or other disease condition isalso contemplated, either before or after tumor resection, or as part ofan art-recognized treatment of the disease condition. Controlled releaseformulations, depot formulations, and infusion pump delivery aresimilarly contemplated.

When used as an anti-cancer agent or an anti-tumor agent, may be orallyor non-orally administered to a human patient in the amount of about0.0007 mg/day to about 7,000 mg/day of the active ingredient, and morepreferably about 0.07 mg/day to about 70 mg/day of the active ingredientat, preferably, one time per day or, less preferably, over two to aboutten times per day. Alternatively and also preferably, the compound maypreferably be administered in the stated amounts continuously by, forexample, an intravenous drip. Thus, for a patient weighing 70 kilograms,the preferred daily dose of the active anti-tumor ingredient would beabout 0.0007 mg/kg/day to about 35 mg/kg/day including 1.0 mg/kg/day and0.5 mg/kg/day, and more preferable, from 0.007 mg/kg/day to about 0.050mg/kg/day, including 0.035 mg/kg/day. Nonetheless, as will be understoodby those of skill in the art, in certain situations it may be necessaryto administer the anti-tumor compound in amounts that excess, or evenfar exceed, the above-stated, preferred dosage range to effectively andaggressively treat particularly advanced or lethal tumors.

When used as an antifungal agent the preferrable amount of thedehydrophenylahistin or its analog effective in the treatment orprevention of a particular fungal pathogen will depend in part on thecharacteristics of the fungus and the extent of infection, and can bedetermined by standard clinical techniques. In vitro or in vivo assaysmay optionally be employed to help identify optimal dosage ranges.Effective doses may be extrapolated from dose-response curves derivedfrom in vitro analysis or preferably from animal models. The precisedosage level should be determined by the attending physician or otherhealth care provider and will depend upon well known factors, includingroute of administration, and the age, body weight, sex and generalhealth of the individual; the nature, severity and clinical stage of theinfection; the use (or not) of concomitant therapies.

The effective dose of the dehydrophenylahistin or its analog willtypically be in the range of about 0.01 to about 50 mg/kgs, preferablyabout 0.1 to about 10 mg/kg of mammalian body weight per day,administered in single or multiple doses. Generally, the compound may beadministered to patients in need of such treatment in a daily dose rangeof about 1 to about 2000 mg per patient.

To formulate the dosage including the compounds disclosed herein as atumor-growth-inhibiting compound, known surface active agents,excipients, smoothing agents, suspension agents and pharmaceuticallyacceptable film-forming substances and coating assistants, and the likemay be used. Preferably alcohols, esters, sulfated aliphatic alcohols,and the like may be used as surface active agents; sucrose, glucose,lactose, starch, crystallized cellulose, mannitol, light anhydroussilicate, magnesium aluminate, magnesium methasilicate aluminate,synthetic aluminum silicate, calcium carbonate, sodium acid carbonate,calcium hydrogen phosphate, calcium carboxymethyl cellulose, and thelike may be used as excipients; magnesium stearate, talc, hardened oiland the like may be used as smoothing agents; coconut oil, olive oil,sesame oil, peanut oil, soya may be used as suspension agents orlubricants; cellulose acetate phthalate as a derivative of acarbohydrate such as cellulose or sugar, or methyiacetate-methacrylatecopolymer as a derivative of polyvinyl may be used as suspension agents;and plasticizers such as ester phthalates and the like may be used assuspension agents. In addition to the foregoing preferred ingredients,sweeteners, fragrances, colorants, preservatives and the like may beadded to the administered formulation of the compound, particularly whenthe compound is to be administered orally.

The compositions disclosed herein in a pharmaceutical compositions mayalso comprise a pharmaceutically acceptable carrier. Such compositionsmay be prepared for storage and for subsequent administration.Acceptable carriers or diluents for therapeutic use are well known inthe pharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).For example, such compositions may be formulated and used as tablets,capsules or solutions for oral administration; suppositories for rectalor vaginal administration; sterile solutions or suspensions forinjectable administration. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution or suspension in liquid prior to injection or infusion, oras emulsions. Suitable excipients include, but are not limited to,saline, dextrose, mannitol, lactose, lecithin, albumin, sodiumglutamate, cysteine hydrochloride, and the like. In addition, ifdesired, the injectable pharmaceutical compositions may contain minoramounts of nontoxic auxiliary substances, such as wetting agents, pHbuffering agents, and the like. If desired, absorption enhancingpreparations (for example, liposomes), may be utilized.

The pharmaceutically effective amount of the composition required as adose will depend on the route of administration, the type of animalbeing treated, and the physical characteristics of the specific animalunder consideration. The dose can be tailored to achieve a desiredeffect, but will depend on such factors as weight, diet, concurrentmedication and other factors which those skilled in the medical artswill recognize.

The products or compositions, as described above, may be used alone orin combination with one another, or in combination with othertherapeutic or diagnostic agents. Specifically, the disclosed compoundsproducts may be used alone or in combination with other chemothepeuticsor bioloigics, including antibodies, for the treatment of cancer, or incombination with other antiinfective for the treatment of fungalinfection. These products or composition can be utilized in vivo or invitro. The useful dosages and the most useful modes of administrationwill vary depending upon the age, weight and animal treated, theparticular compounds employed, and the specific use for which thesecomposition or compositions are employed. The magnitude of a dose in themanagement or treatment for a particular disorder will vary with theseverity of the condition to be treated and to the route ofadministration, and depending on the disease conditions and theirseverity, the compositions may be formulated and administered eithersystemically or locally. A variety of techniques for formulation andadministration may be found in Remington's Pharmaceutical Sciences, 18thed., Mack Publishing Co., Easton, Pa. (1990).

To formulate the compounds of Formula (I), preferably syntheticallyproduced according to the methods disclosed herein, as a cell cycleinhibitor, a tumor-growth-inhibiting, or an antifungal compound, knownsurface active agents, excipients, smoothing agents, suspension agentsand pharmaceutically acceptable film-forming substances and coatingassistants, and the like may be used. Preferably alcohols, esters,sulfated aliphatic alcohols, and the like may be used as surface activeagents; sucrose, glucose, lactose, starch, crystallized cellulose,mannitol, light anhydrous silicate, magnesium aluminate, magnesiummethasilicate aluminate, synthetic aluminum silicate, calcium carbonate,sodium acid carbonate, calcium hydrogen phosphate, calcium carboxymethylcellulose, and the like may be used as excipients; magnesium stearate,talc, hardened oil and the like may be used as smoothing agents; coconutoil, olive oil, sesame oil, peanut oil, soya may be used as suspensionagents or lubricants; cellulose acetate phthalate as a derivative of acarbohydrate such as cellulose or sugar, or methyiacetate-methacrylatecopolymer as a derivative of polyvinyl may be used as suspension agents;and plasticizers such as ester phthalates and the like may be used assuspension agents. In addition to the foregoing preferred ingredients,sweeteners, fragrances, colorants, preservatives and the like may beadded to the administered formulation of the compound produced by themethod, particularly when the compound is to be administered orally.

The cell cycle inhibitors, the antitumor agents, and the antifungalagents that may be produced by the method may be orally or non-orallyadministered to a human patient in the amount of about 0.001 mg/kg/dayto about 10,000 mg/kg/day of the active ingredient, and more preferablyabout 0.1 mg/kg/day to about 100 mg/kg/day of the active ingredient at,preferably, once every three days on a cyclic basis, once oevery otherday, one time per day, twice per day, or less preferably, over two toabout ten times per day. Alternatively and also preferably, the compoundproduced by the method may preferably be administered in the statedamounts continuously by, for example, an intravenous drip. Thus, for theexample of a patient weighing 70 kilograms, the preferred daily dose ofthe active anti-tumor ingredient would be about 0.07 mg/day to about 700grams/day, and more preferable, 7 mg/day to about 7 grams/day.Nonetheless, as will be understood by those of skill in the art, incertain situations it may be necessary to administer the anti-tumorcompound produced by the method in amounts that excess, or even farexceed, the above-stated, preferred dosage range to effectively andaggressively treat particularly advanced or lethal tumors.

In the case of using the cell cycle inhibitor produced by methods as abiochemical test reagent, the compound produced by methods of theinvention inhibits the progression of the cell cycle when it isdissolved in an organic solvent or hydrous organic solvent and it isdirectly applied to any of various cultured cell systems. Usable organicsolvents include, for example, methanol, methylsulfoxide, and the like.The formulation can, for example, be a powder, granular or other solidinhibitor, or a liquid inhibitor prepared using an organic solvent or ahydrous organic solvent. While a preferred concentration of the compoundproduced by the method of the invention for use as a cell cycleinhibitor is generally in the range of about 1 to about 100 μg/ml, themost appropriate use amount varies depending on the type of culturedcell system and the purpose of use, as will be appreciated by persons ofordinary skill in the art. Also, in certain applications it may benecessary or preferred to persons of ordinary skill in the art to use anamount outside the foregoing range.

From a pharmaceutical perspective, certain embodiments provide methodsfor preventing or treating fungal infections and/or a pathogenic fungusin a subject, involve administering to the subject a compositionincluding a dehydrophenylahistin or its analog, for example,administering the dehydrophenylahistin or its analog in an amount andmanner which provides the intended antifungal effect.

Other embodiments include the treatment or prevention of infection in apatient by a pathogenic fungus such as those listed above or referred tobelow.

Another embodiment relates to the treatment or prevention of infectionin a patient by a pathogenic fungus which is resistant to one or moreother antifungal agents, especially an agent other thandehydrophenylahistin or its analog, including e.g. amphotericin B oranalogs or derivatives thereof (including 14(s)-hydroxyamphotericin Bmethyl ester, the hydrazide of amphotericin B with1-amino-4-methylpiperazine, and other derivatives) or other polyenemacrolide antibiotics, including, e.g., nystatin, candicidin, pimaricinand natamycin; flucytosine; griseofulvin; echinocandins oraureobasidins, including naturally occurring and semi-synthetic analogs;dihydrobenzo[a]napthacenequinones; nucleoside peptide antifungalsincluding the polyoxins and nikkomycins; allylamines such as naftifineand other squalene epoxidease inhibitors; and azoles, imidazoles andtriazoles such as, e.g., clotrimazole, miconazole, ketoconazole,econazole, butoconazole, oxiconazole, terconazole, itraconazole orfluconazole and the like. For additional conventional antifungal agentsand new agents under deveopment, see e.g. Turner and Rodriguez, 1996Current Pharmaceutical Design, 2:209-224. Another embodiment involvesthe treatment or prevention of infection in a patient by a pathogenicfungus in cases in which the patient is allergic to, otherwiseintolerant of, or nonresponsive to one or more other antifungal agentsor in whom the use of other antifungal agents is otherwisecontra-indicated. Those other antifungal agents include, among others,those antifungal agents disclosed above and elsewhere herein.

In the foregoing methods for treatment or prevention, adehydrophenylahistin or its analog, is administered to the subject in aneffective antifungal amount.

Other embodiments relate to the treatment or prevention of infection bya pathogenic fungus in a patient by administration of adehydrophenylahistin or its analog, in conjunction with theadministration of one or more other antifungal agents, including forexample, any of the previously mentioned agents or types of agents (e.g.in combination with treatment with amphotericin B, preferably in a lipidor liposome formulation; an azole or triazole such as fluconazole, forexample; an aureobasidin; dihydrobenzo[a]napthacenequinone; or anechinocardin) as well as with a different dehydrophenylahistin or itsanalog.

The dehydrophenylahistin or its analog may be administered before, afteror at the same time the other antifungal agent is administered. Incertain embodiments, the combination therapy will permit the use ofreduced amounts of one or both antifungal components, relative to theamount used if used alone.

Still other embodiments relate to administration of adehydrophenylahistin or its analog to a subject for the treatment orprevention of infection by a pathogenic fungus, where the subject isimmunosuppressed or immunocompromised, e.g. as the result of geneticdisorder, disease such as diabetes or HIV or other infection,chemotherapy or radiation treatment for cancer or other disease, ordrug- or otherwise induced immunosuppression in connection with tissueor organ transplantation or the treatment of an autoimmune disorder.Where the patient is being or will be treated with an immunosuppressiveagent, e.g., in connection with a tissue or organ transplantation, adehydrophenylahistin or its analog may be co-administered with theimmunosuppressive agent(s) to treat or prevent a pathogenic fungalinfection.

Another aspect of this invention is the treatment or prevention ofinfection by a pathogenic fungus in a patient infected, or suspected ofbeing infected, with HIV, by administration of an antifungaldehydrophenylahistin or its analog, in conjunction with theadministration of one or more anti-HIV therapeutics (including e.g. HIVprotease inhibitors, reverse transcriptase inhibitors or anti-viralagents). The dehydrophenylahistin or its analog may be administeredbefore, after or at the same time as administration of the anti-HIVagent(s).

Another aspect of this invention is the treatment or prevention ofinfection by a pathogenic fungus in a patient by administration of anantifungal dehydrophenylahistin or its analog, in conjunction with theadministration of one or more other antibiotic compounds, especially oneor more antibacterial agents, preferably in an effective amount andregiment to treat or prevent bacterial infection. Again, thedehydrophenylahistin or its analog may be administered before, after orat the same time as administration of the other agent(s).

Pathogenic fungal infections which may be treated or prevented by thedisclosed methods include, among others, Aspergillosis, includinginvasive pulmonary aspergillosis; Blastomycosis, including profound orrapidly progressive infections and blastomycosis in the central nervoussystem; Candidiasis, including retrograde candidiasis of the urinarytract, e.g. in patients with kidney stones, urinary tract obstruction,renal transplantation or poorly controlled diabetes mellitus;Coccidioidomycosis, including chronic disease which does not respondwell to other chemotherapy; Cryptococcosis; Histopolasmosis;Mucormycosis, including e.g. craniofacial mucormycosis and pulmonarymucormycosis; Paracoccidioidomycosis; and Sporotrichosis. It should benoted that administration of a composition comprising an antifungalamount of one or more dehydrophenylahistin or its analogs may beparticularly useful for treating or preventing a pathogenic fungalinfection in a mammalian subject where the fungus is resistant to one ormore other antifungal therapies, or where the use of one or more otherantifungal therapies is contraindicated, e.g., as mentioned above.

Antifungal pharmaceutical compositions containing at least oneantifungal dehydrophenylahistin or its analog, are also provided for usein practicing the disclosed methods. Those pharmaceutical compositionsmay be packaged together with an appropriate package insert containing,inter alia, directions and information relating to their antifungal use.Pharmaceutical compositions are also provided which contain one or moredehydrophenylahistin or its analog together with a second antifungalagent.

Methods of Treating Fungal Infections

Certain embodiments disclosed herein relate to methods for treating orpreventing a pathogenic fungal infection, including for exampleAspergillosis, including invasive pulmonary aspergillosis;Blastomycosis, including profound or rapidly progressive infections andblastomycosis in the central nervous system; Candidiasis, includingretrograde candidiasis of the urinary tract, e.g. in patients withkidney stones, urinary tract obstruction, renal transplantaion or poorlycontrolled diabetes mellitus; Coccidioidomycosis, including chronicdisease which does not respond well to other chemotherapy;Cryptococcosis; Histopolasmosis; Mucormycosis, including e.g.craniofacial mucormycosis and pulmonary mucormycosis;Paracoccidioidomycosis; and Sporotrichosis. The methods may involveadministering at least one antifungal dehydrophenylahistin or itsanalog, as described above, to a human subject such that the fungalinfection is treated or prevented. In certain embodiments thedehydrophenylahistin or its analog may be administered in conjunctionwith administration of one or more non-dehydrophenylahistin or itsanalog antifungal agents such as amphotericin B, or an imidazole ortriazole agent such as those mentioned previously.

The pathogenic fungal infection may be topical, e.g., caused by, amongother organisms, species of Candida, Trichophyton, Microsporum orEpiderinophyton or mucosal, e.g., caused by Candida albicans (e.g.thrush and vaginal candidiasis). The infection may be systemic, e.g.,caused by Candida albicans, Cryptococcus neoformans, Aspergillusfumigatus, Coccidiodes, Paracocciciodes, Histoplasma or Blastomyces spp.The infection may also involve eumycotic mycetoma, chromoblastomycosis,cryptococcal meningitits or phycomycosis.

Further embodiments relate to methods for treating or preventing apathogenic fungal infection selected from the group consisting ofCandida spp. including C. albicans, C. tropicalis, C. kefyr, C. kruseiand C. galbrata; Aspergillus spp. including A. fumigatus and A. flavus;Cryptococcus neoibrmans; Blastomyces spp. including Blastomycesdermatitidis; Pneumocvstis carinii; Coccidioides immitis; Basidiobolusranarum; Conidiobolus spp.; Histoplasma capsulatum; Rhizopus spp.including R. oryzae and R. microsporus; Cunninghamella spp.; Rhizoniucorspp.; Paracoccidioides brasiliensis; Pseudallescheria boydii;Rhinosporidium seeberi; and Sporothrix schenckii. Again, the method mayinvolve administering a non-immunosuppressive antifungaldehydrophenylahistin or its analog to a patient in need thereof suchthat the fungal infection is treated or prevented without inducing anuntoward immunosuppressive effect.

Further embodiments relate to methods for treating or preventing apathogenic fungal infection which is resistant to other antifungaltherapy, including pathogenic fungal infections which are resistant toone or more antifungal agents mentioned elsewhere herein such asamphotericin B, flucytosine, one of the imidazoles or triazoles(including e.g. fluconazole, ketoconazole, itraconazole and the otherpreviously mentioned examples). The methods may involve administering tothe patient one or more antifungal dehydrophenylahistin or its analog,in an amount and dosing regimen such that a fungal infection resistantto another antifungal therapy in the subject is treated or prevented.

Further embodiments relate to methods for treating or preventing apathogenic fungal infection in a patient who is allergic to, intollerantof or not responsive to another antifungal therapy or in whom the use ofother antifungal agents is otherwise contra-indicated, including one ormore other antifungal agents mentioned elsewhere herein such asamphotericin B, flucytosine, one of the imidazoles or triazoles(including e.g. fluconazole, ketoconazole, itraconazole and the otherpreviously mentioned examples). The methods may involve administering tosuch patient one or more antifungal dehydrophenylahistin or its analog,in an amount such that a fungal infection is treated or prevented.

Packaged Dehydrophenylahistin or its Analogs

Certain embodiments relate to packaged dehydrophenylahistin or itsanalogs, preferably packaged nonimmunosuppressive antifungaldehydrophenylahistin or its analogs, which term is intended to includeat least one dehydrophenylahistin or its analog, as described above,packaged with instructions for administering the dehydrophenylahistin orits analog(s) as an antifungal agent without causing a untowardimmunosuppressive effects within a human subject. In some embodiments,the non-immunosuppressive antifungal dehydrophenylahistin or its analogis a member of one of the preferred subsets of compounds describedabove. The dehydrophenylahistin or its analog can be packaged alone withthe instructions or can be packaged with another dehydrophenylahistin orits analog, rapamycin or another ingredient or additive, e.g., one ormore of the ingredients of the pharmaceutical compositions. The packagecan contain one or more containers filled with one or more of theingredients of the pharmaceutical compositions. Optionally associatedwith such container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceutical or biological products, which notice reflects approval bythe agency of manufacture, use or sale for human administration.

The following non-limiting examples are meant to describe the preferredmethods using certain preferred embodiments. Variations in the detailsof the particular methods employed and in the precise chemicalcompositions obtained will undoubtedly be appreciated by those of skillin the art.

EXAMPLE 1

A. Synthesis of Dehydrophenylahistin

Dehydrophenylahistin was synthesized by condensation according to thefollowing basic reaction scheme, as shown in FIG. 1:

N,N′-diacetyl-2,5-piperazinedione

25.0 g of global 2,5-piperazinedione 1 [2,5-piperazinedione (AldrichG640-6), 25.0 g, 0.218 mol] in 100 mL of acetic anhydride (Ac₂O) wasmixed with sodium acetate (NaOAc) (17.96 g, 0.0218 mol). The mixture washeated at 110° C. for 8 h using a double coiled condenser under an Aratmosphere. After Ac₂O was removed by evaporation, the residue wasdissolved in AcOEt, washed with 10% citric acid, 10% NaHCO₃ andsaturated NaCl (three times each), dried over Na₂SO₄, and concentratedin vacuo. The residue was triturated with ether to form a solid. Thissolid was recrystallized from EtOAc with ether-hexane to afford 26.4 g(61%) of N,N′-diacetyl-2,5-piperazinedione 1.

1-Acetyl-3-{(Z)-1-[5-(1,1-dimethyl-2-propenyl)-1H-4-imidazolyl]methylidene}]-2,5-piperazinedione2

To a solution of 5-(1,1-dimethyl-2-propenyl)imidazole-4-carboxaldehyde(100 mg, 0.609 mmol) in DMF (2 mL) was added compound 1 (241 mg, 1.22mmol) and the solution was repeatedly evacuated in a short time toremove oxygen and flushed with Ar, followed by the addition of Cs₂CO₃(198 mg, 0.609 mmol) and the evacuation-flushing process was repeatedagain. The resultant mixture was stirred for 5 h at room temperature.After the solvent was removed by evaporation, the residue was dissolvedin the mixture of EtOAc and 10% Na₂CO₃, and the organic phase was washedwith 10% Na₂CO₃ again and saturated NaCl for three times, dried overNa₂SO₄ and concentrated in vacuo. The residual oil was purified bycolumn chromatography on silica using CHCl₃—MeOH (100:0 to 50:1) as aneluant to give 60 mg (33%) of a pale yellow solid 2.

Dehydrophenylahistin

To a solution of 2 (30 mg, 0.099 mmol) in DMF (0.8 mL) was addedbenzaldehyde (51 μL, 0.496 mmol, 5 eq) and the solution was repeatedlyevacuated in a short time to remove oxygen and flushed with Ar, followedby the addition of Cs₂CO₃ (53 mg, 0.149 mmol, 1.5 eq) and theevacuation-flushing process was repeated again. The resultant mixturewas heated for 2.5 h at 80° C. (The temperature must be increasedslowly. Rapid heating increases the production of E-isomer at thebenzylidene moiety.) After the solvent was removed by evaporation, theresidue was dissolved in EtOAc, washed with water for two times andsaturated NaCl for three times, dried over Na₂SO₄ and concentrated invacuo. On TLC using CHCl₃—MeOH (10:1), you can observe a spot withbright green-yellow luminescence at 365 nm UV. The purity of this crudeproduct was more than 75% from HPLC analysis. The resulting residue wasdissolved in 90% MeOH aq and applied to reverse-phase HPLC column(YMC-Pack, ODS-AM, 20×250 mm) and eluted using a linear gradient from 70to 74% MeOH in water over 16 min at a flow rate of 12 mL/min, and thedesired fraction was collected and concentrated by evaporation to give a19.7 mg (60%) of yellow colored dehydrophenylahistin. The HPLC profileof the synthetic crude dehydrophenylahistin is depicted in FIG. 2.

In the purification of dehydrophenylahistin, as shown in FIG. 4, a majorpeak was the desired Z-form compound of dehydrophenylahistin. Theformation of an E-isomer was observed as a minor component (about 10%),which was eluted as a more polar peak than Z-isomer. As other minorpeaks, the reduced Z- and E-compounds, in which the dimethylallyl partof dehydrophenylahistin was reduced, was also observed. The formation ofthese reduced compounds was due to the aldehyde 2 with a reducedimpurity, which was generated during the reduction of with DIBAL-H andwas not separated in the subsequent process.

These minor compounds could be removed by preparative HPLC purification,afforded dehydrophenylahistin with the Z-configuration at thebenzylidene part in a 60% yield (20% yield in two steps) with more than95% purity. The compounds with E-configuration at the imidazole side ofthe diketopiperazine ring was not observed in this HPLC chart,suggesting that the first reaction from compound 1 to 3 in FIG. 1 isZ-selective.

B. Chemical Characteristics:

The above dehydrophenylahistin compound is a pale yellow solid. Itsstructure is confirmed by standard NMR analyses.

EXAMPLE 2 Synthesis and Physical Characterization oftBu-dehydrophenylahistin Derivatives

Structural derivatives of dehydrophenylahistin were synthesizedaccording to the following reaction schemes to producetBu-dehydrophenylahistin. Synthesis by Route A (see FIG. 1) is similarin certain respects to the synthesis of the dehydrophenylahistinsynthesized as in Example 1.

Route A:

N,N′-diacethyl-2,5-piperazinedione 1 was prepared as in Example 1.

1)1-Acetyl-3-{(Z)-1-[5-tert-butyl-1H-4-imidazolyl]methylidene}]-2,5-piperazinedione(16)

To a solution of 5-tert-butylimidazole-4-carboxaldehyde 15 (3.02 g,19.8.

mmol) in DMF (30 mL) was added compound 1 (5.89 g, 29.72 mmol) and thesolution was repeatedly evacuated in a short time to remove oxygen andflushed with Ar, followed by the addition of Cs₂CO₃ (9.7 g, 29.72 mmol)and the evacuation-flushing process was repeated again. The resultantmixture was stirred for 5 h at room temperature. After the solvent wasremoved by evaporation, the residue was dissolved in the mixture ofEtOAc and 10% Na₂CO₃, and the organic phase was washed with 10% Na₂CO₃again and saturated NaCl for three times, dried over Na₂SO₄ andconcentrated in vacuo. The residual oil was purified by columnchromatography on silica using CHCl₃—MeOH (100:0 to 50:1) as an eluantto give 1.90 g (33%) of a pale yellow solid 16. ¹H NMR (270 MHz, CDCl₃)δ 12.14 (d, br-s, 1H), 9.22 (br-s, 1H), 7.57 (s, 1H), 7.18, (s, 1H),4.47 (s, 2H), 2.65 (s, 3H), 1.47 (s, 9H).

2) t-Bu-dehydrophenylahistin

To a solution of1-Acetyl-3-{(Z)-1-[5-tert-butyl-1H-4-imidazolyl]methylidene}]-2,5-piperazinedione(16) (11

mg, 0.038 mmol) in DMF (1.0 mL) was added benzaldehyde (19 μL, 0.19mmol, 5 eq) and the solution was repeatedly evacuated in a short time toremove oxygen and flushed with Ar, followed by the addition of Cs₂CO₃(43 mg, 0.132 mmol, 3.5 eq) and the evacuation-flushing process wasrepeated again. The resultant mixture was heated for 2.5 h at 80° C.After the solvent was removed by evaporation, the residue was dissolvedin EtOAc, washed with water for two times and saturated NaCl for threetimes, dried over Na₂SO₄ and concentrated in vacuo. The resultingresidue was dissolved in 90% MeOH aq and applied to reverse-phase HPLCcolumn (YMC-Pack, ODS-AM, 20×250 mm) and eluted using a linear gradientfrom 70 to 74% MeOH in water over 16 min at a flow rate of 12 mL/min,and the desired fraction was collected and concentrated by evaporationto give a 6.4 mg (50%) of yellow coloredtert-butyl-dehydrophenylahistin. ¹H NMR (270 MHz, CDCl₃) δ 12.34 br-s,1H), 9.18 (br-s, 1H), 8.09 (s, 1H), 7.59 (s, 1H), 7.31-7.49 (m, 5H),7.01 s, 2H), 1.46 (s, 9H).

The dehydrophenylahistin reaction to produce tBu-dehydrophenylahistin isidentical to Example 1.

The total yield of the tBu-dehydrophenylahistin recovered was 16.5%.

Route B:

N,N′-diacethyl-2,5-piperazinedione 1 was prepared as in Example 1.

1)1-Acetyl-3-[(Z)-benzylidene1]-2,5-piperazinedione (17)

To a solution of benzaldehyde 4 (0.54 g, 5.05. mmol) in DMF (5 mL) wasadded compound 1 (2.0 g, 10.1 mmol) and the solution was repeatedlyevacuated in a short time to remove oxygen and flushed with Ar, followedby the addition of Cs₂CO₃ (1.65 g, 5.05 mmol) and theevacuation-flushing process was repeated again. The resultant mixturewas stirred for 3.5 h at room temperature. After the solvent was removedby evaporation, the residue was dissolved in the mixture of EtOAc and10% Na₂CO₃, and the organic phase was washed with 10% Na₂CO₃ again andsaturated NaCl for three times, dried over Na₂SO₄ and concentrated invacuo. The residual solid was recrystalized from MeOH-ether to obtain aoff-white solid of 17; yield 1.95 g (79%).

2) t-Bu-dehydrophenylahistin

To a solution of 1-Acetyl-3-[(Z)-benzylidene1]-2,5-piperazinedione (17)(48 mg, 0.197 mmol) in DMF (1.0 mL) was added5-tert-butylimidazole-4-carboxaldehyde

15 (30 mg, 0.197 mmol) and the solution was repeatedly evacuated in ashort time to remove oxygen and flushed with Ar, followed by theaddition of Cs₂CO₃ (96 mg, 0.296 mmol) and the evacuation-flushingprocess was repeated again. The resultant mixture was heated for 14 h at80° C. After the solvent was removed by evaporation, the residue wasdissolved in EtOAc, washed with water for two times and saturated NaClfor three times, dried over Na₂SO₄ and concentrated in vacuo. Theresulting residue was dissolved in 90% MeOH aq and applied toreverse-phase HPLC column (YMC-Pack, ODS-AM, 20×250 mm) and eluted usinga linear gradient from 70 to 74% MeOH in water over 16 min at a flowrate of 12 mL/min, and the desired fraction was collected andconcentrated by evaporation to give a 0.8 mg (1.2%) of yellow coloredtert-butyl-dehydrophenylahistin.

The total yield of the tBu-dehydrophenylahistin recovered was 0.9%.

The HPLC profile of the crude synthetic tBu-dehyrophenylahistin fromRoute A and from Route B is depicted in FIG. 4.

Two other tBu-dehydrophenylahistin derivatives were synthesizedaccording to the method of Route A. In the synthesis of the additionaltBu-dehydrophenylahistin derivatives, modifications to the benzaldehydecompound 4 were made.

FIG. 4 illustrates the similarities of the HPLC profiles (Column:YMC-Pack ODS-AM (20×250 mm); Gradient: 65% to 75% in a methanol-watersystem for 20 min, then 10 min in a 100% methanol system; Flow rate: 12mL/min; O.D. 230 run) from the synthesized dehydrophenylahistin ofExample 1 (FIG. 2) and the above exemplified tBu-dehydrophenylahistincompound produced by Route A.

The sequence of introduction of the aldehydes is a relevant to the yieldand is therefore aspect of the synthesis. An analogue ofdehydrophenylahistin was sythesized, as a control or model, wherein thedimethylallyl group was changed to the tert-butyl group with a similarsteric hindrance at the 5-position of the imidazole ring.

The synthesis of this “tert-butyl (tBu)-dehydrophenylahistin” using“Route A” was as shown above: Particularly, the sequence of introductionof the aldehyde exactly follows the dehydrophenylahistin synthesis, andexhibited a total yield of 16.5% tBu-dehydrophenylahistin. This yieldwas similar to that of dehydrophenylahistin (20%). Using “Route B”,where the sequence of introduction of the aldehydes is opposite that ofRoute “A” for the dehydrophenylahistin synthesis, only a trace amount ofthe desired tBu-dehydroPLH was obtained with a total yield of 0.9%,although in the introduction of first benzaldehyde 4 gave a 76% yield ofthe intermnediate compound 17. This result indicated that it may bedifficult to introduce the highly bulky imidazole-4-carboxaldehydes 15with a substituting group having a quaternary-carbon on the adjacent5-position at the imidazole ring into the intermediate compound 17,suggesting that the sequence for introduction of aldehydes is animportant aspect for obtaining a high yield of dehydrophenylahistin oran analog of dehydrophenylahistin employing the synthesis disclosedherein.

From the HPLC analysis of the final crude products, as shown in FIG. 4,a very high content of tBu-dehydrophenylahistin and small amount ofby-product formations were observed in the crude sample of Route A(left). However, a relatively smaller amount of the desiredtBu-dehydrophenylahistin and several other by-products were observed inthe sample obtained using Route B (right).

EXAMPLE 3 Alternative, Larger-Scale Synthesis of Dehydrophenylahistinand Analogs Synthesis of3-Z-Benzylidene-6-[5″-(1,1-dimethylallyl)-]H-imidazol-4″-Z-ylmethylene]-piperazine-2,5-dione[Dehydrophenylahistin] (1)

Reagents: a) LDA, CH₃CHO; b) Tos-Cl, pyridine; c) DBU; d) NaOH; e)C₂Cl₂O₂; f) KOOCCH₂COOEt, BuLi; g) SO₂Cl₂; h) H₂NCHO, H₂O; i) LiAlH₄; j)MnO₂; k) 1,4-diacetyl-piperazine-2,5-dione, Cs₂CO₃; l) benzaldehyde,Cs₂CO₃.

3-Hydroxy-2,2-dimethyl-butyric acid methyl ester

A solution of LDA in heptane/THF/ethylbenzene (2 M, 196 ml, 0.39 mol)was added under argon to a solution of methyl isobutyrate (45 ml, 0.39mol) in THF (270 ml) at −60° and the resultant mixture was stirred for30 min. A solution of acetaldehyde (27 ml, 0.48 mol) in THF (45 ml),precooled to −60°, was added slowly and the resulting solution stirredfor a further 30 min. Saturated ammonium chloride (50 ml) was added andthe solution was allowed to warm to room temperature. The reactionmixture was extracted with ethyl acetate, and the extracts were washedwith HCl (2 M), sodium bicarbonate, then brine. The organic layer wasdried over magnesium sulfate, filtered, then evaporated to give a clearoil (52.6 g). Distillation 76-82°/30 mmHg gave pure3-hydroxy-2,2-dimethyl-butyric acid methyl ester (42.3 g, 74%). (Burk etal., J. Am. Chem. Soc., 117:4423-4424 (1995)).

¹H NMR (400 MHz, CDCl₃) δ 1.15 (d, J=6.2 Hz, 3H); 1.17 (s, 6H); 2.66 (d,J=6.2 Hz, 1H, —OH); 3.71 (s, 3H, —OMe); 3.87 (app quintet, J=6.4 Hz, 1H,H3).

2,2-Dimethyl-3-(toluene-4-sulfonyloxy)-butyric acid methyl ester

To a cooled (0°) solution of 3-hydroxy-2,2-dimethyl-butyric acid methylester (52.0 g, 0.36 mol) in pyridine (100 ml) was added gradually,p-toluene sulfonyl chloride (69.0 g, 0.36 mol). The mixture was allowedto warm to room temperature and was stirred for 60 h. The reaction wasagain cooled in ice and was acidified by addition of HCl (2 M). Theresultant solution was extracted with ethyl acetate, the extracts werewashed with HCl, then brine, dried and evaporated to give an oil whichformed a white precipitate upon standing. This mixture was dissolved inthe minimum amount of ethyl acetate and then light petroleum was addedto afford a white precipitate which was collected and washed with morelight petroleum. The filtrate was partially evaporated and a second cropof crystals was collected and added to the first to afford2,2-dimethyl-3-(toluene-4-sulfonyloxy)-butyric acid methyl ester (81.2g, 76%).

¹H NMR (400 MHz, CDCl₃) δ 1.12 (s, 3H); 1.13 (s, 3H); 1.24 (d, J=6.4 Hz,3H); 2.45 (s, 3H, -PhMe) 3.58 (s, 3H, —OMe); 4.94 (quartet, J=6.4 Hz,1H, H3), 7.33 (d, J=8.0 Hz, 2H), 7.78 (d, J=8.0 Hz, 2H).

Evaporation of the final filtrate afforded additional crude2,2-dimethyl-3-(toluene-4-sulfonyloxy)-butyric acid methyl ester (19.0g, 18%).

2,2-Dimethyl-but-3-enoic acid methyl ester

A solution of 2,2-dimethyl-3-(toluene-4-sulfonyloxy)-butyric acid methylester (18.06 g, 0.06 mol) in DBU (15 ml) was heated at 140-160° for 3.5h. The mixture was allowed to cool to room temperature and was thendiluted with ether. The mixture was washed with HCl (1 M), sodiumbicarbonate, then brine. The ethereal layer was dried and partiallyevaporated to give a concentrated solution of 2,2-dimethyl-but-3-enoicacid methyl ester (10 g). (Savu and Katzenellenbogen, J. Org. Chem,46:239-250 (1981)). Further evaporation was avoided due to thevolatility of the product (bp 102°). (Tsaconas et al., Aust. J. Chem.,53:435-437 (2000)).

¹H NMR (400 NMz, CDCl₃) δ 1.31 (s, 6H); 3.68 (s, 3H); 5.06 (d, J =17.1Hz, 1H, —CH═CH₂); 5.11 (d, J=10.7 Hz, 1H, —CH═CH₂); 6.03 (dd, J=17.1,10.7 Hz, 1H, —CH═CH₂).

2,2-Dimethyl-but-3-enoic acid

The above ethereal solution of 2,2-dimethyl-but-3-enoic acid methylester (10 g) was diluted with ethanol (25 ml), sodium hydroxide (4 M, 22ml) was added and the mixture was stirred overnight. The solution waspartially evaporated to remove the ethanol and the resultant mixture wasadded to HCl (1M, 100 ml). The product was extracted with ethyl acetateand the extracts were dried and evaporated to give2,2-dimethyl-but-3-enoic acid (6.01 g, 88% 2 steps). (Hayashi et al., J.Org. Chem., 65:8402-8405 (2000).

¹H NMR (400 MHz, CDCl₃) δ 1.33 (s, 6H); 5.11 (d, J=10.8 Hz, 1H,—CH═CH₂); 5.15 (d, J =17.2 Hz, 1H, —CH═CH₂); 6.05 (dd, J =17.2, 10.8 Hz,1H, —CH═CH₂)

Monoethyl hydrogen malonate (Wierenga and Skulnick, “Aliphatic andAromatic β-keto Esters from Monoethyl Malonate: Ethyl 2-Butyrylacetate,”Organic Syntheses Collective Volume 7, 213).

Ethyl potassium malonate (25.0 g, 0.15 mol) was suspended in water (15.6ml) and cooled in an ice bath. Concentrated HCl (12.5 ml) was addeddropwise over 30 min, then the mixture was stirred for a further 10 min.The precipitate was filtered, then washed twice with ether. The filtratewas separated and the aqueous phase was extracted with ether. Thecombined ethereal solutions were dried (MgSO₄) and evaporated to afford,as an oil, monoethyl hydrogen malonate (19.2 g, 99%) which was driedunder vacuum overnight (or 50°/1 mm for 1 h) prior to use.

4,4-Dimethyl-3-oxo-hex-5-enoic acid ethyl ester

Oxalyl chloride (3.83 ml, 43.9 mmol) was added dropwise to a cooled (0°)solution of 2,2-dimethyl-but-3-enoic acid (5.0 g, 43.9 mmol) and DMF (1drop) in anhydrous dichloromethane (25 ml). The mixture was stirred for1 h at 0°, then for 16 h at room temperature. Fractional distillation(121°/760 mmHg) afforded 2,2-dimethyl-but-3-enoyl chloride (4.1 g, 71%).

Monoethyl hydrogen malonate (7.2 g, 0.05 mol) and bipyridyl (fewmilligrams) were dissolved in THF (90 ml) and the system was flushedwith nitrogen. The solution was cooled to −70°, then BuLi (2.5 M inhexanes, 37 ml, 0.09 mol) was added. After the addition of only ˜10 mlof BuLi the solution turned pink and additional THF (15 ml) was requiredto enable magnetic stirring. The cooling bath was removed and theremaining BuLi was added, the temperature was allowed to reach −10°,upon which the solution turned colorless. The mixture was again cooledto −60° and a solution of 2,2-dimethyl-but-3-enoyl chloride (4.1 g, 0.03mol) in THF (12 ml) was added dropwise. After addition was complete themixture was allowed to warm to 0° and stir for 3 h, then it was added toa 1:1 mixture of ether/1M HCl (260 ml) at 0° and stirred for a further1.5 h. The organic layer was removed, washed with HCl (1 M), sodiumbicarbonate solution, brine then dried and evaporated to give4,4-dimethyl-3-oxo-hex-5-enoic acid ethyl ester (5.6 g, 98%). (Hayashiet al., J. Org. Chem., 65:8402-8405 (2000). Distillation with aKugelrohr oven (160°/1 mmHg) afforded pure material.

¹H NMR (400 MHz, CDCl₃) δ 1.26 (s, 6H); 1.27 (t, J =6.9 Hz, 3H,—CH₂CH₃); 3.51 (s, 2H); 4.18 (q, J=6.9 Hz, 2H, —CH₂CH₃); 5.20 (d, J=17.7Hz, 1H, —CH═CH₂); 5.21 (d, J=9.6 Hz, 1H, —CH═CH₂); 5.89 (dd, J=17.7, 9.6Hz, 1H, —CH═CH₂).

2-Chloro-4,4-dimethyl-3-oxo-hex-5-enoic acid ethyl ester

Sulfuryl chloride (0.84 ml, 10.4 mmol) was added to a cooled (0°)solution of 4,4-dimethyl-3-oxo-hex-5-enoic acid ethyl ester (1.83 g,9.93 mmol) in chloroform (7 ml). The resulting mixture was allowed towarm to room temperature and stir for 30 min, after which it was heatedunder reflux for 2 h. After cooling to room temperature the reactionmixture was diluted with chloroform, then was washed with sodiumbicarbonate, water then brine. The organic phase was dried andevaporated to afford, as a brown oil,2-chloro-4,4-dimethyl-3-oxo-hex-5-enoic acid ethyl ester (2.01 g, 93%).(Hayashi et al., J. Org. Chem., 65:8402-8405 (2000).

¹H NMR (400 MHz, CDCl₃) δ 1.28 (t, J=7.0 Hz, 3H, —CH₂CH₃); 1.33 (s, 3H);1.34 (s, 3H); 4.24 (q, J=7.0 Hz, 2H, —CH₂CH₃); 5.19 (s, 1H; 5.28 (d,J=16.9 Hz, 1H, —CH═CH₂); 5.29 (d, J=10.9 Hz, 1H, —CH═CH₂); 5.96 (dd,J=16.9, 10.9 Hz, 1H, —CH═CH₂).

LC/MS t_(R)=8.45 (219.3 [M(Cl³⁷)+H]⁺ min.

This material was reacted without further purification.

5-(1,1-Dimethyl-allyl)-3H-imidazole-4-carboxylic acid ethyl ester

A suspension of 2-chloro-4,4-dimethyl-3-oxo-hex-5-enoic acid ethyl ester(19.4 g, 0.09 mol) and water (1.94 ml, 0.11 mol) in formamide (36.8 ml)was shaken briefly, then dispensed into 15×18 ml vials. The vials weresealed and heated at 150° for 5 h. After cooling to room temperature,the vials' contents were combined and extracted exhaustively withchloroform. The extracts were dried and evaporated to afford aconcentrated formamide solution (14.7 g). This was added to a silicacolumn (7 cm diameter, 11 cm height) packed in 1% MeOH/1% Et₃N inchloroform. Elution of the column with 2 L of this mixture followed by 2L of 2% MeOH/1% Et₃N in chloroform afforded, in the early fractions, acompound suspected of being 5-(1,1-dimethyl-allyl)-oxazole-4-carboxylicacid ethyl ester (1.23 g. 7%).

HPLC (214 nm) t_(R)=8.68 (50.4%) min.

¹H NMR (400 MHz, CDCl₃) δ 1.40 (t, J=7.2 Hz, 3H, —CH₂CH₃); 1.54 (s, 6H);4.38 (t, J=7.2 Hz, 2H, —CH₂CH₃); 5.03 (d, J=17.4 Hz, 1H, —CH═CH₂); 5.02(d, J=10.4 Hz, 1H, —CH═CH₂); 6.26 (dd, J=17.4, 10.4 Hz, 1H, —CH═CH₂);7.83 (s, 1H).

LCMS t_(R)=8.00 (210.1 [M+H]⁺, 361.1 [2M+H]⁺) min.

Recovered from later fractions was the desired5-(1,1-dimethyl-allyl)-3H-imidazole-4-carboxylic acid ethyl ester (3.13g, 17%). (Hayashi et al., J. Org. Chem., 65:8402-8405 (2000)).

HPLC (214 nm) t_(R)=5.52 (96.0%) min.

¹H NMR (400 MHz, CDCl₃) δ 1.38 (t, J=7.0 Hz, 3H); 1.57 (s, 6H); 4.35 (q,J =7.0 Hz, 2H); 5.04-5.14 (m, 2H, —CH═CH₂); 6.28 (dd, J =18.0, 10.4 Hz,1H, —CH═CH₂); 7.52 (s, 1H).

LC/MS t_(R)=5.30 (209.1 [M+H]⁺, 417.2 [2M+H]⁺) min.

Additional 5-(1,1-dimethyl-allyl)-3H-imidazole-4-carboxylic acid ethylester was also recovered from the column (3.59 g, 19%) which was oflower purity but still sufficient for further reaction.

Another byproduct isolated from a similar reaction (smaller scale) byfurther elution of the column with 5% MeOH/1% Et₃N in chloroform was acompound suspected of being5-(1,1-dimethyl-allyl)-3H-imidazole-4-carboxylic acid (0.27 g, 9%).

HPLC (245 nm) t_(R)=5.14 (68.9%) min.

¹H NMR (400 MHz, CD₃OD) δ 1.45 (s, 6H); 4.97 (d, J=10.6 Hz, 1H,—CH═CH₂); 5.01 (d, J=17.7 Hz, 1H, —CH═CH₂); 6.28 (dd, J=17.7, 10.6 Hz,1H, —CH═CH₂); 7.68 (s, 1H).

LCMS t_(R)=4.72 (181.0 [M+H]⁺, 361.1 [2M+H]⁺) min.

[5-(1,1-Dimethyl-allyl)-3H-imidazol-4-yl]-methanol

A solution of 5-(1,1-dimethyl-allyl)-3H-imidazole-4-carboxylic acidethyl ester (3.13 g, 15.0 mmol) in THF (60 ml) was added dropwise to asuspension of lithium aluminium hydride (95% suspension, 1.00 g, 25.0mmol) in THF (40 ml) and the mixture was stirred at room temperature for4 h. Water was added until the evolution of gas ceased, the mixture wasstirred for 10 min, then was filtered through a sintered funnel. Theprecipitate was washed with THF, then with methanol, the filtrate andwashings were combined, evaporated, then freeze-dried to afford[5-(1,1-dimethyl-allyl)-3H-imidazol-4-yl]-methanol (2.56 g, 102%).Residual water was removed by azeotroping with chloroform prior tofurther reaction. (See Hayashi et al., J. Org. Chem., 65:8402-8405(2000)).

HPLC (240 nm) t_(R)=3.94 (56.8%) min.

¹H NMR (400 MHz, CD₃OD) δ 1.43 (s, 6H); 4.57 (s, 2H); 5.01 (d, J=10.5Hz, 1H, —CH═CH₂); 5.03 (d, J=17.7 Hz, 1H, —CH═CH₂); 6.10 (dd, J=17.7,10.5 Hz, 1H, —CH═CH₂); 7.46 (s, 1H).

LC/MS t_(R)=3.77 (167.3 [M+H]⁺) min.

5-(1,1-Dimethyl-allyl)-3H-imidazole-4-carbaldehyde

Manganese dioxide (20 g, 0.23 mol) was added to a solution of[5-(1,1-dimethyl-allyl)-3H-imidazol-4-yl]-methanol (2.56 g, 0.02 mol) inacetone (300 ml) and the resulting mixture was stirred at roomtemperature for 5 h. The mixture was filtered through filter paper andthe residue was washed with acetone. The filtrate and washings werecombined and evaporated to afford5-(1,1-dimethyl-allyl)-3H-imidazole-4-carbaldehyde (1.82 g, 51%).(Hayashi et al., J. Org. Chem., 65:8402-8405 (2000)).

HPLC (240 nm) t_(R)=4.08 (91.5%) min.

¹H NMR (400 MHz, CDCl₃) δ 1.56 (s, 6H); 5.16 (d, J=10.6 Hz, 1H,—CH═CH₂); 5.19 (d, J=17.3 Hz, 1H, CH═CH₂); 6.22 (dd, J=17.3, 10.6 Hz,1H, —CH═CH₂); 7.75 (s, 1H), 10.02 (s, 1H, HCO).

LC/MS t_(R)=3.75 (165.2 [M+H]⁺) min.

1-Acetyl-3-[5′-(1,1-dimethyl-allyl)-1H-imidazol-4′-Z-ylmethylene]-piperazine-2,5-dione

To a solution of 5-(1,1-dimethyl-allyl)-3H-imidazole-4-carbaldehyde(1.78 g, 0.01 mol) in DMF (35 ml) was added1,4-diacetyl-piperazine-2,5-dione (8.59 g, 0.04 mol) and the mixture wasevacuated, then flushed with argon. The evacuation-flushing process wasrepeated a further two times, then cesium carbonate (3.53 g, 0.01 mol)was added. The evacuation-flushing process was repeated a further threetimes, then the resultant mixture was heated at 45° for 5 h. Thereaction mixture was partially evaporated (heating under high vacuum)until a small volume remained and the resultant solution was addeddropwise to ice-water (50 ml). The yellow precipitate was collected,washed with water, then freeze-dried to afford1-acetyl-3-[5′-(1,1-dimethyl-allyl)-1H-imidazol-4′-ylmethylene]-piperazine-2,5-dione(1.18 g, 36%). (Hayashi, Personal Communication (2001)).

HPLC (214 nm) t_(R)=6.01 (72.6%) min.

¹H NMR (400 MHz, CDCl₃) δ 1.53 (s, 6H); 2.64 (s, 3H); 4.47 (s, 2H); 5.19(d, J=17.3 Hz, 1H, —CH═CH₂); 5.23 (d, J=10.7 Hz, 1H, —CH═CH₂); 6.06 (dd,J=17.3, 10.7 Hz, 1H, —CH═CH₂); 7.16 (s, 1H), 7.59 (s, 1H), 9.47 (bs,1H); 12.11 (bs, 1H) [observed ˜2% 1,4-diacetyl-piperazine-2,5-dionecontamination δ 2.59 (s, 6H); 4.60 (s, 4H).]

LC/MS t_(R)=6.65 (303.3 [M+H]⁺, 605.5 [2M+H]⁺) min. (n.b. differentsystem used).

3-Z-Benzylidene-6-[5″-(1,1-dimethylallyl)-1H-imidazol-4″-Z-ylmethylene]-piperazine-2,5-dione

To a solution of1-acetyl-3-[5′-(1,1-dimethyl-allyl)-1H-imidazol-4′-ylmethylene]-piperazine-2,5-dione(2.91 g, 9.62 mmol) in DMF (70 ml) was added benzaldehyde (4.89 ml, 48.1mmol) and the solution was evacuated, then flushed with Argon. Theevacuation-flushing process was repeated a further two times, thencesium carbonate (4.70 g, 14.4 mmol) was added. The evacuation-flushingprocess was repeated a further three times, then the resultant mixturewas heated under the temperature gradient ad shown below.

After a total time of 5 h the reaction was allowed to cool to roomtemperature and the mixture was added to ice-cold water (500 ml). Theprecipitate was collected, washed with water (300 ml), then freeze-driedto afford a yellow solid (2.80 g). This material was dissolved inchloroform (250 ml) filtered through filter paper and evaporated toazeotrope remaining water. The residual yellow precipitate (2.70 g, HPLC(214nm) t_(R)=7.26 (93.6%) min.) was partially dissolved in chloroform(20 ml), the suspension was sonicated for 5 min, then the solid wascollected and air dried to afford3-Z-benzylidene-6-[5″-(1,1-dimethylallyl)-1H-imidazol-4″-Z-ylmethylene]-piperazine-2,5-dione(1.82 g, 54%) (Hayashi, Personal Communication (2001)), m.p. 239-240°(dec.).

HPLC (214 nm) t_(R)=6.80 (1.92) min, 7.33 (95.01%).

¹H NMR (400 MHz, CDCl₃) δ 1.53 (s, 6H); 5.18 (d, J=17.6 Hz, 1H,—CH═CH₂); 5.21 (d, J=11.0 Hz, 1H, —CH═CH₂); 6.06 (dd, J=17.6, 11.0 Hz,1H, —CH═CH₂); 6.99 (s, 1H, —C—C═CH); 7.00 (s, 1H, —C—C═CH); 7.30-7.50(m, 5×ArH); 7.60 (s, H2″); 8.07 (bs, NH); 9.31 (bs, NH); 12.30 (bs, NH).

LC/MS t_(R)=6.22 (349.3 [M+H]⁺, E isomer), 6.73 (349.5 [M+H]⁺, 697.4[2M+H]⁺, Z isomer) min.

ESMS m/z 349.5 [M+H]⁺, 390.3 [M+CH₄CN]⁺.

Evaporation of the chloroform solution gave additional3-Z-benzylidene-6-[5″-(1,1-dimethylallyl)-1H-imidazol-4″-Z-ylmethylene]-piperazine-2,5-dione(0.76 g, 29%).

HPLC (214 nm) t_(R)=7.29 (84.5%) min.

3-E-Benzylidene-6-[5″-(1,1-dimethylallyl)-1H-imidazol-4″-Z-ylmethylene]-piperazine-2,5-dione

Preparative HPLC purification of a crude sample of material synthesizedas above afforded the geometric isomer3-E-Benzylidene-6-[5″-(1,1-dimethylallyl)-1H-imidazol-4″-Z-ylmethylene]-piperazine-2,5-dione(1.7 mg).

HPLC (214 nm) t_(R)=6.75 (87.79) min.

¹H NMR (400 MHz, CDCl₃) δ 1.52 (s, 6H); 5.19 (d, J =20.8 Hz, 1H,CH═CH₂); 5.22 (d, J=14.0 Hz, 1H, CH═CH₂); 6.05 (dd, J=18.0, 10.4 Hz, 1H,CH═CH₂); 6.33 (s, 1H, C—C═CH); 6.90-7.65 (m, 7H).

ESMS m/z 349.5 [M+H]⁺, 390.4 [M+CH₄CN]⁺.

Synthesis of3-Z-Benzylidene-6-(5″-tert-butyl-1H-imidazol-4″-Z-ylmethylene)-piperazine-2,5-dione(2)

Reagents: g) SO₂Cl₂; h) H₂NCHO, H₂O; I)LiAlH₄; j) MnO₂; k)1,4-diacetyl-piperazine-2,5-dione, Cs₂CO₃; l) benzaldehyde, Cs₂CO₃.

2-Chloro-4,4-dimethyl-3-oxo-pentanoic acid ethyl ester

Sulfuryl chloride (14.0 ml, 0.17 mol) was added to a cooled (0°)solution of ethyl pivaloylacetate (27.17 g, 0.16 mol) in chloroform (100ml). The resulting mixture was allowed to warm to room temperature andwas stirred for 30 min, after which it was heated under reflux for 2.5h. After cooling to room temperature, the reaction mixture was dilutedwith chloroform, then washed with sodium bicarbonate, water then brine.

The organic phase was dried and evaporated to afford, as a clear oil,2-chloro-4,4-dimethyl-3-oxo-pentanoic acid ethyl ester (33.1 g, 102%).(Durant et al., “Aminoalkylimidazoles and Process for their Production.”Patent No. GB1341375 (Great Britain, 1973)).

HPLC (214 nm) t_(R)=8.80 (92.9%) min.

¹H NMR (400 MHz, CDCl₃) δ 1.27 (s, 9H); 1.29 (t, J=7.2 Hz, 3H); 4.27 (q,J=7.2 Hz, 2H); 5.22 (s, 1H).

¹³C NMR (100 MHz, CDCl₃) δ 13.8, 26.3, 45.1, 54.5, 62.9, 165.1, 203.6.

5-tert-Butyl-3H-imidazole-4-carboxylic acid ethyl ester

A solution of 2-chloro-4,4-dimethyl-3-oxo-pentanoic acid ethyl ester(25.0 g, 0.12 mol) in formamide (47.5 ml) and water (2.5 ml) was shaken,then dispensed into 15×8 ml vials. All vials were sealed and then heatedat 150° for 3.5 h. The vials were allowed to cool to room temperature,then water (20 ml) was added and the mixture was exhaustively extractedwith chloroform. The chloroform was removed to give a concentratedformamide solution (22.2 g) which was added to a flash silica column (6cm diameter, 12 cm height) packed in 1% MeOH/1% Et₃N in chloroform.Elution of the column with 2.5 L of this mixture followed by 1 L of 2%MeOH/1% Et₃N in chloroform gave, in the early fractions, a productsuspected of being 5-tert-butyl-oxazole-4-carboxylic acid ethyl ester(6.3 g, 26%).

HPLC (214 nm) t_(R)=8.77 min.

¹H NMR (400 MHz, CDCl₃) δ 1.41 (t, J=7.2 Hz, 3H); 1.43 (s, 9H); 4.40 (q,J=7.2 Hz, 2H); 7.81 (s, 1H).

¹³C NMR (100 MHz, CDCl₃) δ 14.1, 28.8, 32.5, 61.3, 136.9, 149.9, 156.4,158.3.

ESMS m/z 198.3 [M+H]⁺, 239.3 [M+CH₄CN]⁺.

LC/MS t_(R)=7.97 (198.1 [M+H]⁺) min.

Recovered from later fractions was5-tert-butyl-3H-imidazole-4-carboxylic acid ethyl ester (6.20 g, 26%).(Durant et al., “Aminoalkylimidazoles and Process for their Production.”Patent No. GB1341375 (Great Britain, 1973)).

HPLC (214 nm) t_(R)=5.41 (93.7%) min.

¹H NMR (400 MHz, CDCl₃) δ 1.38 (t, J=7.0 Hz, 3H); 1.47 (s, 9H); 4.36 (q,J=7.2 Hz, 2H); 7.54 (s, 1H).

¹³C NMR (100 MHz, CDCl₃) δ 13.7, 28.8, 32.0, 59.8, 124.2, 133.3, 149.2,162.6.

ESMS m/z 197.3 [M+H]⁺, 238.3 [M+CH₄CN]⁺.

Further elution of the column with 1 L of 5% MeOh/1% Et₃N gave acompound suspected of being 5-tert-butyl-3H-imidazole-4-carboxylic acid(0.50 g, 2%).

HPLC (245 nm) t_(R)=4.68 (83.1%) min.

¹H NMR (400 MHz, CD₃OD) δ 1.36 (s, 9H); 7.69 (s, 1H).

¹H NMR (400 MHz, CDCl₃) δ 1.37 (s, 9H); 7.74 (s, 1H).

¹H NMR (400 MHz, CD₃SO) δ 1.28 (s, 9H); 7.68 (s, 1H).

ESMS m/z 169.2 [M+H]⁺, 210.4 [M+CH₄CN]⁺.

(5-tert-Butyl-3H-imidazol-4-yl)-methanol

A solution of 5-tert-butyl-3-imidazole-4-carboxylic acid ethyl ester(3.30 g, 16.8 mmol) in THF (60 ml) was added dropwise to a suspension oflithium aluminium hydride (95% suspension, 0.89 g, 22.2 mmol) in THF (40ml) and the mixture was stirred at room temperature for 3 h. Water wasadded until the evolution of gas ceased, the mixture was stirred for 10min, then was filtered through a sintered funnel. The precipitate waswashed with THF, then with methanol, the filtrate and washings werecombined and evaporated. The residue was freeze-dried overnight toafford, as a white solid (5-tert-butyl-3H-imidazol-4-yl)-methanol (2.71g, 105%). (Durant et al., “Aminoalkylimidazoles and Process for theirProduction.” Patent No. GB1341375 (Great Britain, 1973)).

HPLC (240 nm) t_(R)=3.70 (67.4%) min.

¹H NMR (400 MHz, CD₃OD) δ 1.36 (s, 9H); 4.62 (s, 2H); 7.43 (s, 1H).

¹³C NMR (100 MHz, CD₃OD) δ 31.1, 33.0, 57.9, 131.4, 133.9, 140.8.

LC/MS t_(R)=3.41 (155.2 [M+H]⁺) min.

This material was used without further purification.

5-tert-Butyl-3H-imidazole-4-carbaldehyde

Manganese dioxide (30 g, 0.35 mol) was added to a heterogeneous solutionof (5-tert-butyl-3H-imidazol-4-yl)-methanol (4.97 g, 0.03 mol) inacetone (700 ml) and the resulting mixture was stirred at roomtemperature for 4 h. The mixture was filtered through a pad of Celiteand the pad was washed with acetone. The filtrate and washings werecombined and evaporated. The residue was triturated with ether toafford, as a colorless solid, 5-tert-butyl-3H-imidazole-4-carbaldehyde(2.50 g, 51%). (Hayashi, Personal Communication (2000)).

HPLC (240 nm) t_(R)=3.71 (89.3%) min.

¹H NMR (400 MHz, CDCl₃) δ 1.48 (s, 9H); 7.67 (s, 1H); 10.06 (s, 1H).

LC/MS t_(R)=3.38 (153.2 [M+H]⁺) min.

Evaporation of the filtrate from the trituration gave additional5-tert-butyl-3H-imidazole-4-carbaldehyde (1.88 g, 38%).

1-Acetyl-3-(5′-tert-butyl-1H-imdazol-4′-Z-ylmethylene)-piperazine-2,5-dione

To a solution of 5-tert-butyl-3H-imidazole-4-carbaldehyde (2.50 g, 164.4mmol) in DMF (50 ml) was added 1,4-diacetyl-piperazine-2,5-dione (6.50g, 32.8 mmol) and the solution was evacuated, then flushed with argon.The evacuation-flushing process was repeated a further two times, thencesium carbonate (5.35 g, 16.4 mmol) was added. The evacuation-flushingprocess was repeated a further three times, then the resultant mixturewas stirred at room temperature for 5 h. The reaction mixture waspartially evaporated (heat and high vacuum) until a small volumeremained and the resultant solution was added dropwise to water (100ml). The yellow precipitate was collected, then freeze-dried to afford1-acetyl-3-(5′-tert-butyl-1H-imidazol-4′-Z-ylmethylene)-piperazine-2,5-dione(2.24 g, 47%). (Hayashi, Personal Communication (2000)).

HPLC (214 nm) t_(R)=5.54 (94.4%) min.

¹H NMR (400 MHz, CDCl₃) δ 1.47 (s, 9H); 2.65 (s, 3H), 4.47 (s, 2H); 7.19(s, 1H); 7.57 (s, 1H), 9.26 (s, 1H), 12.14 (s, 1H).

¹³C NMR (100 MHz, CDCl₃+CD₃OD) δ 27.3, 30.8, 32.1, 46.5, 110.0, 123.2,131.4, 133.2, 141.7, 160.7, 162.8, 173.0.

LC/MS t_(R)=5.16 (291.2 [M+H]⁺, 581.6 [2M+H]⁺) min.

3-Z-Benzylidene-6-(5″-tert-butyl-1H-imidazol-4″-Z-ylmethylene)-piperazine-2,5-dione

To a solution of1-acetyl-3-(5′-tert-butyl-1H-imidazol-4′-Z-ylmethylene)-piperazine-2,5-dione(2.43 g, 8.37 mmol) in DMF (55 ml) was added benzaldehyde (4.26 ml, 41.9mmol) and the solution was evacuated, then flushed with nitrogen. Theevacuation-flushing process was repeated a further two times, thencesium carbonate (4.09 g, 12.6 mmol) was added. The evacuation-flushingprocess was repeated a further three times, then the resultant mixturewas heated under the temperature gradient as shown below. After a totaltime of 5 h the reaction was allowed to cool to room temperature and themixture was added to ice-cold water (400 ml). The precipitate wascollected, washed with water, then freeze-dried to afford a yellow solid(2.57 g, HPLC (214 nm) t_(R)=6.83 (83.1%) min.). This material wasdissolved in chloroform (100 ml) and evaporated to azeotrope remainingwater, resulting in a brown oil. This was dissolved in chloroform (20ml) and cooled in ice. After 90 min the yellow precipitate was collectedand air-dried to afford3-Z-benzylidene-6-(5″-tert-butyl-1H-imidazol-4″-Z-ylmethylene)-piperazine-2,5-dione(1.59 g, 56%). (Hayashi, Personal Communication (2000)).

HPLC (214 nm) t_(R)=6.38 (2.1%), 6.80 (95.2) min.

¹H NMR (400 MHz, CDCl₃) δ 1.46 (s, 9H); 7.01 (s, 1H, —C—C═CH); 7.03 (s,1H, —C—C═CH); 7.30-7.50 (m, 5H, Ar); 7.60 (s, 1H); 8.09 (bs, NH); 9.51(bs, NH); 12.40 (bs, NH).

LC/MS t_(R)=5.84 (337.4 [M+H]⁺, E isomer), 6.25 (337.4 [M+H]⁺, 673.4[2M+H]⁺, Z isomer) min.

ESMS m/z 337.3 [M+H]⁺, 378.1 [M+CH₄CN]⁺.

Evaporation of the chloroform solution gave additional3-Z-benzylidene-6-(5″-tert-butyl-1H-imidazol-4″-Z-ylmethylene)-piperazine-2,5-dione(0.82 g, 29%). HPLC (214 nm) t_(R)=6.82 (70.6%) min.

General Experimental

Sodium bicarbonate refers to a 5% solution.

Organic solvents were dried over sodium sulfate unless otherwise stated.

Analytical Conditions

NMR Conditions

¹H NMR (400 MHz) analysis was performed on a Varian Inova Unity 400 MHzNMR machine. Samples were run in deuterated chloroform containing 0.1%TMS (unless otherwise specified). Chemical shifts (ppm) are referencedrelative to TMS (0.00 ppm) or CH₃OH at 3.30 ppm for samples run CD₃OD.Coupling constants are expressed in hertz (Hz).

Analytical HPLC Conditions

System 6 conditions:

RP-HPLC was done on a Rainin Microsorb-MV C18 (5 μm, 100 Å) 50×4.6 mmcolumn.

Buffer A: 0.1% aqueous TFA.

Buffer B: 0.1% TFA in 90% aqueous MeCN.

Gradient: 0-100% Buffer B over 11 min.

Flow rate: 1.5 mL/min.

LCMS Conditions

LCMS were run on a Perkin-Elmer Sciex API-100 instrument.

LC conditions:

Reverse Phase HPLC analysis.

Column: Monitor 5 μm C18 50×4.6 mm.

Solvent A: 0.1% TFA in water.

Solvent B: 0.085% TFA in 90% aqueous MeCN.

Gradient: 0-100% B over 11.0 min.

Flow rate: 1.5 mL/min.

Wavelength: 214 nm.

MS conditions:

Ion Source: Ionspray.

Detection: Ion counting.

Flow rate to the mass spectrometer: 300 μL/min after split from column(1.5 mL/min).

ESMS Conditions

ESMS was done on a Perkin Elmer/Sciex-API III LC/MS/MS using anelectrospray inlet.

Solvent: 0.1% AcOH in 60% aqueous MeCN.

Flow rate: 25 μL/min.

Ionspray: 5000 V.

Orifice plate: 55 V.

Acquisition time: 2.30 min.

Scan range: 100-1000 amu/z.

Scan step size: 0.2 amu/z.

Preparative RP-HPLC Purification Conditions

Reverse phase HPLC purification was carried out using Nebula with theWaters XterraMS column (19×50 mm, 5 μm, C18) using the followingconditions:

Solvent A: 0.1% aqueous TFA.

Solvent B: 0.1% TFA in 90% aqueous MeCN.

Gradient: 5-95% B over 4 min.

Flow rate: 20 mL/min.

Wavelength: 214 nm.

Abbreviations are as follows: br s: broad singlet; BuLi: n-butyllithium; d: doublet; DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene; ESMS:electrospray mass spectrometry; HCl: hydrochloric acid; HPLC: highperformance liquid chromatography; LCMS: liquid chromatography massspectrometry; LD: lithium diisopropylamide; M+: molecular ion; m:multiplet; MeCN: acetonitrile; M: mass spectrometry; MW: molecularweight; NMR: nuclear magnetic resonance; q: quartet; s: singlet;triplet; t_(R): retention time; TFA: trifluoroacetic acid; THF:tetrahydrofuranDetailed Procedure for the Synthesis of Dehydrophenylahistin

1-Acetyl-3-{(Z)-1-[5-(1,1-dimethyl-2-propenyl)-1H-4-imidazolyl]methylidene}]-2,5-piperazinedione(2)

To a solution of 5-(1,1-dimethyl-2-propenyl)imidazole-4-carboxaldehyde(100 mg, 0.609 mmol) in DMF (2 mL) was added compound 1 (241 mg, 1.22mmol) and the solution was repeatedly evacuated in a short time toremove oxygen and flushed with Ar, followed by the addition of Cs₂CO₃(198 mg. 0.609 mmol) and the evacuation-flushing process was repeatedagain. The removal of oxygen is prefered because such removal isbelieved to decrease oxidation of alpha-carbon at the position 6 of thediketopiperazine ring. The resultant mixture was stirred for 5 h at roomtemperature. After the solvent was removed by evaporation, the residuewas dissolved in the mixture of EtOAc and 10% Na₂CO₃, and the organicphase was washed with 10% Na₂CO₃ again and saturated NaCl for threetimes, dried over Na₂CO₃ and concentrated in vacuo. The residual oil waspurified by column chromatography on silica using CHCl₃—MeOH (100:0 to50:1) as an eluant to give 60 mg (33%) of a pale yellow solid 2.

Dehydrophenylahistin

To a solution of 2 (30 mg, 0.099 mmol) in DMF (0.8 mL) was addedbenzaldehyde (51 μL, 0.496 mmol, 5 eq) and the solution was repeatedlyevacuated in a short time to remove oxygen and flushed with Ar, followedby the addition of Cs₂CO₃ (53 mg, 0.149 mmol, 1.5 eq) and theevacuation-flushing process was repeated again. The resultant mixturewas heated for 2.5 h at 80° C. (The temperature must be increasedslowly. Rapid heating increases the production of E-isomer at thebenzylidene moiety.) After the solvent was removed by evaporation, theresidue was dissolved in EtOAc, washed with water for two times andsaturated NaCl for three times, dried over Na₂SO₄ and concentrated invacuo. On TLC using CHCl₃—MeOH (10:1), you can observe a spot withbright green-yellow luminescence at 365 nm UV. The purity of this crudeproduct was more than 75% from HPLC analysis. The resulting residue wasdissolved in 90% MeOH aq and applied to reverse-phase HPLC column(YMC-Pack, ODS-AM, 20×250 mm) and eluted using a linear gradient from 70to 74% MeOH in water over 16 min at a flow rate of 12 mL/min, and thedesired fraction was collected and concentrated by evaporation to give a19.7 mg (60%), although the yields are not optimized for each step, ofyellow colored dehydrophenylahistin.

EXAMPLE 4 Biological Characteristics of Dehydrophenylahistin andDehydrophenylahistin Analogs

A. Biological Evaluation

The biological characteristics of synthesized tBu-dehydrophenylahistinand dehydrophenylahistin were evaluated in both HT29 human colon cells,and PC-3 prostatic adenocarcinoma cells.

HT-29 (ATCC HTB-38) a human colorectal adenocarcinoma was maintained inMcCoy's complete medium (McCoy's SA medium with L-glutamine and 25 mMHEPES supplemented with 10% FBS, 1 mM Na pyruvate, 1×NEAA, 2 mML-glutamine, and Pen/Strep at 100 IU/ml and 100 μg/ml, respectively).PC-3 (ATCC CRL-1435), a human prostate adenocarcinoma, was maintained inF12K complete medium (F12K medium supplemented with 10% FBS; 2mMGlutamine; 1% HEPES; and Pen/Strep at 100 IU/ml and 100 μg/ml,respectively). Cell lines were cultured at 37° C., 5% CO₂ in a 95%humidified incubator.

For tumor cytotoxicity assays HT-29 or PC-3 cells were seeded at 5,000cells/well in 90 μl complete media into a Corning 3904 black-walled,clear-bottom tissue culture plate and the plate were incubated overnightto allow cells to establish and enter log phase growth. 20 mM stocksolutions of dehydrophenylahistin and tBu-dehydrophenylahistin wereprepared in 100% DMSO and stored at −20° C. 10× concentrated serialdilutions of the two compounds were prepared in appropriate culturemedium for final concentrations ranging from 20×10⁻⁵ M to 20×10⁻¹⁰ M.Ten μl volumes of the 10× serial dilutions were added to the test wellsin triplicate and the plates returned to the incubator for 48 hours. Thefinal concentration of DMSO was 0.25% in all samples.

Following 48 hours of drug exposure 10 μl of 0.2 mg/ml resazurin(obtained from Sigma-Aldrich Chemical Co.) in Mg²⁺, Ca²⁺ free PBS wasadded to each well and the plates were returned to the incubator for 3-4hours. The plates were removed and resazurin fluorescence was measuredusing 530 nm excitation and 590 nm emission filters in a Fusionfluorimeter (Packard Instruments). Resazurin dye without cells was usedto determine the background, which was subtracted from the data for allexperimental wells. The data were analyzed using Prism software(GraphPad Software). The data were normalized to the average of thecells treated with media only (100% cell growth) and EC₅₀ values weredetermined using a standard sigmoidal dose response curve fittingalgorithm.

As indicated in Table 1 below, tBu-dehydrophenylahistin demonstratesabout a 4-times greater cytotoxic activity in comparison withdehydrophenylahistin. TABLE 1 Cytotoxic Effect of dehydrophenylahistinand derivative.

Dehydrophenylahistin

tBu-dehydrophenylahistin EC₅₀ (nM) cell ΔPLH tBu-ΔPLH HT29 48 13 PC-35.4 1.0B. Structure and Activity Study of Dehydrophenylahistin Derivatives

The cytotoxic effects of phenylahistin, dehydrophenylahistin and variousderivatives of dehydrophenylahistin were examined in P388 murineleukemia cells, HT-29 human colon cells, and PC-3 prostaticadenocarcinoma cells.

As explained above, HT-29 a human colorectal adenocarcinoma wasmaintained in McCoy's complete medium (McCoy's 5A medium withL-glutamine and 25 mM HEPES supplemented with 10% FBS, 1 mM Na pyruvate,1×NEAA, 2 mM L-glutamine, and Pen/Strep at 100 IU/ml and 100 μg/ml,respectively). PC-3, a human prostate adenocarcinoma, was maintained inF12K complete medium (F12K medium supplemented with 10% FBS; 2 mMGlutamine; 1% HEPES; and Pen/Strep at 100 IU/ml and 100 μg/ml,respectively). Cell lines were cultured at 37° C., 5% CO₂ in a 95%humidified incubator.

For tumor cytotoxicity assays HT-29 or PC-3 cells were seeded at 5,000cells/well in 90 μl complete media into a Coming 3904 black-walled,clear-bottom tissue culture plates and the plates were incubatedovernight to allow cells to establish and enter log phase growth. 20 mMstock solutions of dehydrophenylahistin and tBu-dehydrophenylahistinwere prepared in 100% DMSO and stored at −20° C. 10× concentrated serialdilutions of the two compounds were prepared in appropriate culturemedium for final concentrations ranging from 20×10⁻⁵ M to 20×10⁻¹⁰ M.Ten μl volumes of the 10× serial dilutions were added to the test wellsin triplicate and the plates returned to the incubator for 48 hours. Thefinal concentration of DMSO was 0.25% in all samples.

Following 48 hours of drug exposure 10 μl of 0.2 mg/ml resazurin(obtained from Sigma-Aldrich Chemical Co.) in Mg²⁺, Ca²⁺ free PBS wasadded to each well and the plates were returned to the incubator for 3-4hours. The plates were removed and resazurin fluorescence was measuredusing 530 nm excitation and 590 nm emission filters in a Fusionfluorimeter (Packard Instruments). Resazurin dye without cells was usedto determine the background, which was subtracted from the data for allexperimental wells. The data were analyzed using Prism software(GraphPad Software). The data were normalized to the average of thecells treated with media only (100% cell growth) and EC₅₀ values weredetermined using a standard sigmoidal dose response curve fittingalgorithm.

EC₅₀ and IC₅₀ values of phenylahistin, dehydrophenylahistin anddehydrophenylahistin derivatives are summarized in Table 2 below. TABLE2 SAR study of phenylahistin or dehydrophenylahistin and ofdehydrophenylahistin derivatives EC₅₀ (nM) IC₅₀ (nM) COMPOUNDS STRUCTUREHT-29 PC-3 P-388 (−)-Phenylahistin

1600 n.t. 833 ± 153 (n = 5) KPU-1 ΔPLH

48 4.7   36 ± 12.8 (n = 5) KPU-2 tBu-ΔPLH

13 1 31.8 ± 5.0 (n = 5) KPU-6 tBu-ΔPLH-p-OMe

>2000 n.t. 9333 ±5457 (n = 3) KPU-8 tBu-ΔPLH-o-OMe

89 315 ± 137 (n = 4) KPU-9 tBu-ΔPLH-M-OMe

31 20.8 ± 68  (n = 4) Colchicine — 208 ± 68  (n = 4)

Modifications to the phenyl ring have a significant effect of thecytotoxic activities. In comparison with the activity oftBu-dehydrophenylahistin (#6), the activity of the methoxy group at themeta-position (KPU-9) exhibited the highest activity than the otherderivatives with an IC₅₀ of 20.8±3.3 nM in P388 cells. The KPU-9derivative also exhibited cytotoxicity in HT-29 cells (EC₅₀ 31 nM).Dehydrophenylahistin, tBu-dehydrophenylahistin (KPU-2) and the KPU-9derivative all exhibited cytotoxicity in P388 cells.

C. Structure and Activity Study of Additional DehydrophenylahistinDerivatives

The cytotoxic effects of phenylahistin, dehydrophenylahistin and variousadditional derivatives of dehydrophenylahistin were examined in HT-29human colon cells and PC-3 prostatic adenocarcinoma cells using themethodology described above. TABLE 3 SAR study of phenylahistin,dehydrophenylahistin and of additional dehydrophenylahistin derivativesSALT EC₅₀ (nM) COMPOUNDS STRUCTURE FORM M.W. HT-29 PC-3(−)-Phenylahistin

— 350.41 1600 n.t. KPU-1 ΔPLH

— 348.40 48  4.7 KPU-2 tBu-ΔPLH

— 336.39 13  1 KPU-6 tBu-ΔPLH-p-OMe

— 366.41 >2000 n.t. KPU-8 tBu-ΔPLH-o-OMe

— 366.41 89 KPU-9 tBu-ΔPLH-m-OMe

— 366.41 31 KPU-14 tBu-ΔPLH-2,3- diOMe

TFA 396.44 510.46 (+TFA) 610  96% KPU-12 tBu-ΔPLH-2,4- diOMe

— 396.44 4980 KPU-10 tBu-ΔPLH-2,5- diOMe

— 396.44 1350 KPU-15 tBu-ΔPLH-2,6- diOMe

TFA 396.44 510.46 (+TFA) 4430  96% KPU-13 tBu-ΔPLH-3,4- diOMe

— 396.44 2130 KPU-16 tBu-ΔPLH-3,5- diOMe

— 396.44 42  82% KPU-11 tBu-ΔPLH-3,4,5- triOMe

— 426.47 >20 μM KPU-17 tBu-ΔPLH-2,3,4- triOMe

TFA 426.47 540.49 (+TFA) 4060  94% KPU-18 tBu-ΔPLH-o-Cl

TFA 370.83 484.86 (+TFA) 42 100% KPU-19 tBu-ΔPLH-m-Cl

TFA 370.83 484.86 (+TFA) 20  98% KPU-20 tBu-ΔPLH-p-Cl

TFA 370.83 484.86 (+TFA) 545 KPU-21 tBu-ΔPLH-2Cl-5- NO₂

TFA 415.83 529.85 (+TFA) 51 100% KPU-22 tBu-ΔPLH-3,4- methylene-dioxy

TFA 380.40 494.92 (+TFA) 82  95% KPU-23 tBU-ΔPLH-2-OH- 3-OMe(o-vanillin)

TFA 382.41 496.44 (+TFA) 5870  86% KPU-24 tBu-ΔPLH- cyclized-3-MeO

TFA 364.40 487.42 (+TFA) 7040 100% KPU-25 tBu-ΔPLH-4- pyridyl

TFA 337.38 565.42 (+2TFA) 544  98% KPU-28 tBu-ΔPLH-2- pyridyl

TFA 337.38 565.42 (+2TFA) >20 μM  99% KPU-26 tBu-ΔPLH-2-furyl

TFA 326.35 440.37 (+TFA) 600  88% KPU-27 tBu-ΔPLH-5-Me- 2-thienyl

TFA 356.44 470.47 (+TFA) 80  97% KPU-29 tBu-ΔPLH-3-Me- 2-thienyl

TFA 356.44 470.47 (+TFA) 44  81%

EXAMPLE 5 Other Dehydrophenylahistin Analogs

A. Modifications for the Synthesis of Dehydrophenylahistin Derivatives

Other derivatives of dehydrophenylahistin are synthesized using theforegoing techniques alone or in conjunction with other well knownorganic synthesis techniques.

Modifications to the diacyldiketopiperazine and the first and secondaldehydes involved in the synthesis method vary according to the desiredderivative to produce. Derivatives are synthesized that:

-   -   A) modify the phenyl ring and/or introduce other aromatic ring        systems,    -   B) alter the position of the aromatic ring,    -   C) alter the imidazole aromatic ring system, and/or    -   D) modify the 5-position on the imidazole ring.

The figure below depicts regions of the dehydrophenylahistin compoundmodified to produce derivatives of dehydrophenylahistin. Non-limitingexamples of modifications are disclosed, and based on this disclosurewould be understood by those of skill in the art.

A 1) Modification of the phenyl ring besed on the structure of knownanti-tubulin compounds Alkyl, Halogen, Alkoxy, Acetyl, Sulfonamide,Amino, Hydroxyl, Nitro, etc.

2) Introduction of other aromatic ring systems

B Position of the aromatic ring

C Change to the other ring systems

D Further modification of the 5-positin on the imidazole ring

Expanding on the above modifications to the dehydrophenylahistincompound, derivatives of the compound may include the followingsubstitutions at the phenyl ring (A): —CF₃, —SO₂NH₂(—SO₂NR₁R₂), —SO₃H,—CONH₂(—CONR₁R₂), —COOH, etc. Other ring systems (C) may also includethe following:

B. Examples of Synthesized Dehydrophenylahistin Derivatives

Additional examples of synthesized dehydrophenylahistin derivatives aredisclosed in the Table 4. TABLE 4 Additional synthesized derivatives ofdehydrophenylahistin SALT COMPOUNDS STRUCTURE FORM M.W. KPU-20tBu-ΔPLH-p-Cl

TFA 370.83 484.86 (+TFA) KPU-30 tBu-ΔPLH-2,3-methylendioxy

TFA 380.40 494.42 (+TFA) KPU-31 tBu-ΔPLH-3-pyridyl

2TFA 337.38 565.42 (+2TFA) KPU-32 tBu-ΔPLH-o-Me

TFA 350.41 464.44 (+TFA) KPU-33 tBu-ΔPLH-3-Me-2-pyridyl

2TFA 351.40 579.45 (+2TFA) KPU-34 tBu-ΔPLH-4-F

TFA 354.38 468.40 (+TFA) KPU-35 tBu-ΔPLH-m-F

TFA 354.38 468.40 (+TFA) KPU-36 tBu-ΔPLH-5-Me-4-im

2TFA 356.42 584.47 (+2TFA) KPU-37 tBu-ΔPLH-o-F

TFA 354.38 468.40 (+TFA) KPU-38 tBu-ΔPLH-m-Me

TFA 350.41 464.44 (+TFA) KPU-39 tBu-ΔPlh-p-Me

TFA 350.41 464.44 (+TFA) KPU-40 tBu-ΔPLH-p-Br

TFA 415.28 529.31 (+TFA) KPU-41 tBu-ΔPLH-m-Br

TFA 415.28 529.31 (+TFA) KPU-42 tBu-ΔPLH-3-thienyl

TFA 342.42 456.44 (+TFA) KPU-43 tBu-ΔPLH-p-CN

TFA 361.40 475.42 (+TFA) KPU-44 tBu-ΔPLH-m-EtO

TFA 380.44 494.46 (+TFA) KPU-45 tBu-ΔPLH-2,4,6-TriOMe

TFA 426.47 540.49 (+TFA) KPU-46 tBu-ΔPLH-o-NO₂

TFA 381.39 495.41 (+TFA) KPU-47 tBu-ΔPLH-m-NO₂

TFA 381.39 495.41 (+TFA) KPU-48 tBu-ΔPLH-p-NO₂

TFA 381.39 495.41 (+TFA) KPU-49 tBu-ΔPLH-m-CN

TFA 361.40 475.42 (+TFA) LPU-50 tBu-ΔPLH-o-Br

TFA 415.28 529.31 (+TFA) KPU-51 tBu-ΔPLH-m-OH

TFA 352.39 466.41 (+TFA) KPU-52 tBu-ΔPLH-2-NO₂-5-Cl

TFA 415.83 529.85 (+TFA) KPU-53 tBu-ΔPLH-o-OH

TFA 352.39 466.41 (+TFA) KPU-54 tBu-ΔPLH-2-OH-5-OMe

TFA 382.41 496.44 (+TFA) KPU-55 tBu-ΔPLH-3-furanyl

TFA 326.35 440.37 (+TFA) KPU-56 tBu-ΔPLH-2-OH-5-Br

TFA 431.28 545.31 (+TFA) KPU-57 ΔPLH-2-OH-4-OMe

TFA 382.41 496.44 (+TFA) KPU-58 tBu-ΔPLH-2-OH-4-OMe

TFA 382.41 496.44 (+TFA) KPU-59 tBu-ΔPLH-2-OH-5-Cl

TFA 386.83 (+TFA) 500.86 KPU-60 tBu-ΔPLH-5-Me-2-furanyl

TFA 340.38 454.40 (+TFA) KPU-61 tBu-ΔPLH-5-Cl-2-thionyl

TFA 376.86 490.88 (+TFA) KPU-62 tBu-ΔPLH-2-thionyl

TFA 342.42 456.44 (+TFA) KPU-63 tBu-ΔPLH-N-Me-2-pyrrole

TFA 339.39 453.42 (+TFA) KPU-64 tBu-ΔPLH-3,5-diCl

TFA 405.27 KPU-65 tBu-ΔPLH-m-CF₃

TFA 404.39 KPU-66 tBu-ΔPLH-1-Naphthalene

TFA 386.44 KPU-67 tBu-ΔPLH-2-Naphthalene

TFA 386.44 KPU-68 TBu-ΔPLH-2,3-diCl

TFA 405.27 KPU-69 TBu-ΔPLH-m-Vinyl

TFA 362.42 KPU-77 TBu-ΔPLH-2-F-5-I

TFA 480.28 KPU-79 tBu-ΔPLH-2-(Methylthio)

TFA 368.45 KPU-80 TBu-ΔPLH-m-OCF₃

TFA 420.38 KPU-81 TBu-ΔPLH-2-F-5-OMe

TFA 384.38 KPU-82 TBu-ΔPLH-4-F-3-OMe

TFA 384.38 KPU-83 TBu-ΔPLH-2-OH-5-tBu

TFA 408.39 KPU-84 TBu-ΔPLH-cyclohexane

TFA 341.39 KPU-86 TBu-ΔPLH-2-Me-3-F

TFA 368.39 KPU-87 TBu-ΔPLH-2-F-5-Me

TFA 368.39 KPU-88 TBu-ΔPLH-2-Cl-6-F

TFA 388.83 KPU-89 TBu-ΔPLH-2,5-di-F

TFA 372.38 KPU-90 TBu-ΔPLH-2,3-di-Me

TFA 364.38 KPU-91 TBu-ΔPLH-2,6-di-Me

TFA 364.38 KPU-92 tBu-ΔPLH-2-NO₂-3-OMe

TFA 411.39 KPU-93 TBu-ΔPLH-2,5-diMe

TFA 364.38 KPU-94 tBu-ΔPLH-2-NH₂-3-OMe

TFA  381.399 KPU-96 TBu-ΔPLH-2-NH₂

TFA 351.41 KPU-97 TBu-ΔPLH-m-NH₂

TFA 351.41C. Evaluation of Dehydrophenylahistin Derivatives

Evaluation of derivatives described above are assessed according to themethods described in Example 3. Additional evaluation of the derivativesare extended to specific activities such as determining the inhibitingeffect on cell proliferation, the effects on a specific cellularmechanism (i.e. microtuble function), effects on cell cycle progression,evaluating in vitro anti-tumor activity against cancer cell lines, etc.Some evaluation method protocols are given below.

1) Cell Proliferation Inhibiting Effect of Dehydrophenylahistin and itsAnalogs

Into each well of a 96-well microtiter plate, 100μl of A-549 cellsderived from human lung cancer prepared to 105 cells/ml in a culturemedium obtained by adding 10% bovine fetus serum to EMEM culture medium(Nissui Seiyaku Co., Ltd.) having antitumor effect against A-549 cellsderived from human lung cancer are placed. Methanol solution of thederivative obtained by the above-listed examples are added to the wellsof the uppermost row, specimens are diluted by the half-log dilutionmethod and added, and the plate is incubated in a carbon dioxide gasincubator at 37° C. for 48 hours. The result is added in lots of 10 μlwith MTT reagent (3-(4,5-dimethyl-2-thiazole)-2,5-diphenyl-2H-tetrabromide)(1 mg/ml PBS), followed by incubation in a carbon dioxide gasincubator at 37° C. for 6 hours. The culture medium is discarded and thecrystal of produced in the cells are dissolved in 100 μl/well ofdimethylsulfoxide. Absorption of 595 nm light is then measured with amicroplate reader. By comparing the light absorptions of the untreatedcells to that of cells treated with a specimen of a known concentration,the specimen concentration that inhibited cell proliferation 50% (IC₅₀)is calculated.

2) Cell Cycle Inhibiting Activity of Dehydrophenylahistin and itsAnalogs

Cell strain A431 is derived from human lung cancer. EMEM culture mediumcontaining 10% bovine fetal serum and 1% MEM nonessential amino acidsolution (SIGMA M2025) is used to incubate A431 cells at 37° C. in anincubator saturated with 5% carbon dioxide gas and water vapor. Therefined specimen of dehydrophenylahistin obtained in by the methodsabove are added to the cells in the log-growth phase and progression ofthe cell cycle is analyzed by flow cytometer and microscopicobservation.

EXAMPLE 6 Structure-Activity Relationship of SynthesizedDehydrophenylahistin (DehydroPLH) Derivatives

1) Overview in Derivative Syntheses

Many, but not all, of the derivatives of dehydroPLH disclosed hereininclude one, two, or three modifications at the phenyl ring (FIG. 5below). The derivatives were synthesized by the methods described above.As shown in Table 5, certain compounds showed more potent cytotoxicactivity than dehydroPLH and tBu-dehydroPLH. The most potent compoundexhibiting an EC50 value of 3 nM was KPU-90. This value was 16-times and4-times higher than that of dehydroPLH and tBu-dehydroPLH, respectively.These derivatives have mono-substitution at the o- or m-position of thephenyl ring with the halogen atoms such as fluorine and chlorine atomsor the methyl, vinyl or methoxy group. Derivatives with substitutions toheteroaryl structures such as the npahthalene, thiophene and furan ringsalso elicited a potent activity. KPU-35, 42, 69, 80 and 81 also showedhigher activity than tBu-dehydroPLH. TABLE 5 Synthetic potent dehydroPLHderivatives Compound Structure EC₅₀ (nM) KPU-9

31 KPU-35

10 KPU-18

42 KPU-19

20 KPU-38

45 KPU-37

21 KPU-41

31 KPU-29

44 KPU-16

42 KPU-32

42 KPU-42

54 KPU-46

44 KPU-44

43 tBu-ΔPLH (KPU-2)

13 KPU-69

16 KPU-80

13 KPU-81

19 KPU-90

3 DehydroPL H (KPU-1)

48

2) Introduction of the Methoxy Groups to the Phenyl Ring

Colchicine recognizes the same binding site on β-tubulin as PLH.Colchicine has four characteristic methoxy groups on its A and B rings.A series of substitutions with the single or multiple methoxy groups wasperformed and the results of cytotoxic activity is shown in Table 6.TABLE 6 Effect of the methoxy group substitution on the proliferation ofHT-29 cells Compound Structure EC50 (nM) DehydroPLH (KPU-1)

48 tBuΔPLH (KPU-2)

13 KPU-8

89 KPU-9

31 KPU-6

6730 KPU-10

1350 KPU-12

4980 KPU-13

2130 KPU-14

610 KPU-15

4430 KPU-16

42 KPU-24

7040

The result demonstrated that substitutions at the m- or o-positionincreased cytotoxic activity against HT-29 cells. KPU-9 and 16 showedhigh activity. The methoxy-derivatives with triple substitution (KPU-11,17 and 45) also showed activity. The structure of KPU-24 was assigned byMASS analysis.

3) Modification with the Electron-Withdrawing Groups

To study more expanded structure-activity relationship on the phenylring, a series of different functional groups were introduced, whichinclude both electron-withdrawing and -donating groups. The result ofcytotoxicity against HT-29 cells is shown in Tables 7 and 8,respectively.

Substitution at the o- or m-position effectively increased activity.These results were well consistent with the case of the methoxy group.TABLE 7 Effect of the electron-withdrawing group on proliferation ofHT-29 cells Compound Structure EC₅₀ (nM) KPU-18

42 KPU-19

20 KPU-20

545 KPU-21

51 KPU-52

110 KPU-37

21 KPU-35

10 KPU-34

466 KPU-50

38 KPU-41

31 KPU-40

623 KPU-46

44 KPU-47

40 KPU-48

>20 μM KPU-49

28 KPU-43

>20 μM

TABLE 8 Effect of the electron-donating group on proliferation of HT-29cells Compound Structure EC₅₀ (nM) KPU-8

89 KPU-9

31 KPU-6

6730 KPU-44

43 KPU-30

477 KPU-22

82 KPU-32

42 KPU-38

45 KPU-39

460 KPU-53

>20 μM KPU-51

617 KPU-23

5870 KPU-58

>20 μM KPU-54

>20 μM KPU-57

>20 μM

The present disclosure is not bound by or limited to any particularscientific theory. Nonethless, it is appreciated that persons of skillin the art may interpret the results presented herein to suggest that arelatively smaller functional group, affecting less steric hinderance,may be preferred to elicit more potent activity, and slightly largegroups such as the ethoxy group (when compared to the methoxy group) orthe Br atom (when compared to the Cl atom) may affect steric hindranceunfavorable to interaction with, for example, the tubulin binding site.Moreover, because the electrical property of these substituents did notaffect the activity, it is suggested that these relatively smallsubstituents do not directly interact with the binding site ofβ-tubulin, but restrict the conformation of dehydroPLH suitable for thebinding. Or, as another possible hypothesis, the hydrophobic propertymay be a more important factor at the binding site for o- or m-positionon β-tubulin, since introduction of the hydrophilic hydroxyl group,which can form the hydrogen bonding as a hydrogen-donor, drasticallydecreased the activity.

As shown in Table 9, the effect of the substituents in the cytotoxicactivity at the o-position may be ordered, as in the case of m-position,as shown in Table 10. The compounds having effective functional groups,which showed higher activity than tBu-dehydroPLH, may also be furthermodified. And since the migration of the stereochemistry from Z to Eunder the visible light irradiation was observed, substituents thatdecrease the electron density in the conjugated double bonds maycontribute to the reduction of Z to E migration by the light, results inmore physicochemically stable structures. Temperature can also effectthis migration.

Modification at two parts of the ring can be prefered for thedevelopment of potent but also biologically stable compounds. The phenylring of phenylahistin is oxidized by cytochrome P-450. Doublemodification that reduce the electron density of the phenyl ring maytherefore be effective to avoid P-450 oxidation. Thus, the combinationof the small electron withdrawing group such as the fluorine atom to theelement that can increase the activity such as —OMe, —Me, —Cl, —F andBr, may result in more potent and biologically stable drug compounds.TABLE 9 Summary of modification at the o-position EC₅₀ CompoundStructure (nM) KPU-2 

48 KPU-8 

89 KPU-37

21 KPU-18

42 KPU-50

38 KPU-46

44 KPU-32

42 KPU-53

>20 μM

TABLE 10 Summary of modification at the m-position Compound StructureEC₅₀ (nM) KPU-2 

48 KPU-9 

31 KPU-35

10 KPU-19

20 KPU-41

31 KPU-47

40 KPU-38

45 KPU-51

617 KPU-49

28 KPU-44

43

4) Substitution of the Phenyl Ring to Aryl-Heterocycles

The phenyl ring may also be replaced by heteroaryl groups. The result ofsuch replacements in terms of the cytotoxic activity are shown in Table11. Since the arylic nitrogen atoms can form a hydrogen bonding with aNH group of the diketopiperazine ring and restrict the conformation ofthe molecule between pyridine and diketopiperazine rings to an uniplanarstructure, the active conformation of dehydroPLH would be required acertain level of dihedral angle formed by the steric repulsion betweenan amide hydrogen atom of the diketopiperazine ring and an o-hydrogenatom of the phenyl ring (FIG. 6). TABLE 11 Effect of the replacementwith the heteroaryl ring on proliferation of HT-29 cells CompoundStructure EC₅₀ (nM) KPU-28

>20 μM KPU-31

96 KPU-25

544 KPU-33

>20 μM KPU-26

600 KPU-60

71 KPU-42

54 KPU-27

80 KPU-29

44 KPU-61

184 KPU-36

2790 KPU-63

105

Replacing the phenyl ring with a smaller furan or thiophene ring, forexample, KPU-29 or -42, exhibited activity. The phenyl ring can bechanged to other aromatic structure while maintaining the potentactivity.

5) Metabolism of Phenylahistin

In the recent his study, (±)-phenylahisitn was treated with rat hepaticmicrosome or human hepatic P450s. In human case at least sevenmetabolites were detected, and two of them, i.e., P1 and P3, were majormetabolites, represented more than 60% of the recovered metabolites.

Since there is no exo-olefin structure in tBu-dehydroPLH, presentsynthesized derivatives have no oxidization like P1 and P4. However,oxidizations such as P3 and P5 are formed during the hepatic metabolism.Various derivatives, which prevent such metabolism, are effective toavoid P450 oxidization at the phenyl ring. The imidazole ring can alsobe modified to avoid the unfavorable oxidation.

6) Physicochemical Stability of DehydroPLH

The physicochemical stability is one of the unfavorable problems ofdehydroPLH. In phenylahistin, since there is no additional olefinstructure at the benzyl part, there is no such problem. However, indehydroPLH, the benzylidene moiety can be easily activated, probablywith the visible light, and the Z to E migration frequently occurs dueto the existence of longer conjugation of the double bond. Thismigration occurred even under normal room light. In the cytotoxic assay,some of the compounds migrate to E-form during the incubation, althoughthis migration probably equilibrates at the 1:1 ratio in the case ofdehydroPLH. This migration can be controled. The Z to E migration isalso known in combretastatin A4, a same type of tubulin inhibitor, and afew studies for improving this problem were reported.

7) Prodrug Synthesis

The E-form may also be used as a prodrug of dehydroPLH or of one or moreof its analogs, including those analogs described herein. One of theundesired properties of anti-tubulin drugs involves its low selectivitybetween tumor and intact tissues, although these drugs belong to one ofthe molecular target therapies. This causes undesired side effects.However, if the compounds functions selectively only in tumor tissues,negative side effects of anti-microtubule drugs can be reduced. Sincethe dehydroPLH (Z-form) can be produced from its E-isomer by visiblelight irradiation, the E-form is administered and photo irradiation isperformed only at the tumor site, then only the tumor is damaged byphoto-produced Z-form and the adverse effect to the intact tissues isreduced.

The E-form can be protected chemically by the addition of a bulky butbiodegradable acyl group, which is introduced into the diketopiperazinering as a prodrug. This acyl group can be cleaved by the protease in thebody. Therefore, the acylated-E-compound is maintained beforeadministration, then after administration it is changed to the realE-form, which can migrate to the bioactive Z-form by the local photoirradiation.

The synthetic scheme of this acyl-E-form of tBu-dehydroPLH is summarizedin FIG. 9.

EXAMPLE 7 Pharmaceutical Formulations of the SynthesizedDehydrophenylahistins

1) Formulations Administered Intravenously, by Drip, Injection, Infusionor the Like

Vials containing 5 g of powdered glucose are each added aseptically with10 mg of a compound synthesized by the method and sealed. After beingcharged with nitrogen, helium or other inert gas, the vials are storedin a cool, dark place. Before use, the contents are dissolved in ethanoland added to 100 ml of a 0.85% physiological salt water solution. Theresultant solution is administered as a method of inhibiting the growthof a cancerous tumor in a human diagnosed as having such a tumor atbetween approximately 10 ml/day to approximately 1000 ml/day,intravenously, by drip, or via a subcutaneous or intraperitonealinjection, as deemed appropriate by those of ordinary skill in the art.

2) Formulation to be Administered Orally or the Like

A mixture obtained by thoroughly blending 1 g of a compound synthesizedby the method, 98 g of lactose and 1 g of hydroxypropyl cellulose isformed into granules by any conventional method. The granules arethoroughly dried and sifted to obtain a granule preparation suitable forpackaging in bottles or by heat sealing. The resultant granulepreparations are orally administered at between approximately 100 ml/dayto approximately 1000 ml/day, depending on the symptoms, as deemedappropriate by those of ordinary skill in the art of treating canceroustumors in humans.

3) Formulation to be Administered Topically

Administration to an individual of an effective amount of the compoundcan also be accomplished topically by administering the compound(s)directly to the affected area of the skin of the individual. For thispurpose, the compound administered or applied is in the form of acomposition including a pharmacologically acceptable topical carrier,such as a gel, an ointment, a lotion, or a cream, which includes,without limitation, such carriers as water, glycerol, alcohol, propyleneglycol, fatty alcohols, triglycerides, fatty acid esters, or mineraloils. Other topical carriers include liquid petroleum, isopropylpalmitate, polyethylene glycol, ethanol (95%), polyoxyethylenemonolaurate (5%) in water, or sodium lauryl sulfate (5%) in water. Othermaterials such as anti-oxidants, humectants, viscosity stabilizers, andsimilar agents may be added as necessary. Percutaneous penetrationenhancers such as Azone may also be included. In addition, in certaininstances, it is expected that the compound may be disposed withindevices placed upon, in, or under the skin. Such devices includepatches, implants, and injections which release the compound into theskin, by either passive or active release mechanisms.

EXAMPLE 8 In Vitro Pharmacology of KPU-2, KPU-35 and t-butylPhenylahistin

The in vitro efficacy studies performed with KPU-2, KPU-35 and t-butylphenylahistin included: A) a panel of six tumor cell lines, B) studiesin multidrug-resistant tumor cells, and C) studies to determine themechanism of action.

A). Study of KPU-2. KPU-35 and t-butyl Phenylahistin in a Panel of SixTumor Cell Lines

The following cell lines (source in parentheses) were used: HT29 (humancolon tumor; ATCC; HTB-38), PC3 (human prostate tumor; ATCC; CRL-1435),MDA-MB-231 (human breast tumor; ATCC; HTB-26), NCI-H292 (human non-smallcell lung tumor; ATCC; CRL-1848), OVCAR-3 (human ovarian tumor; ATCC;HTB-161), B16-F10 (murine melanoma; ATCC; CRL-6475) and CCD-27sk (normalhuman fibroblast; ATCC; CRL-1475). Cells were maintained at subconfluentdensities in their respective culture media.

Cytotoxicity assays were performed as described above in Example 4,using Resazurin fluorescence as an indicator of cell viability.

The disclosed compounds are effective agents against a variety ofdifferent and distinct tumor cell lines. Specifically, for example,KPU-2 and KPU-35 were most effective on the HT-29 tumor cell line, bothin terms of potency (active in the low nanomolar range) and efficacy(most responsive in terms of the maximum cytotoxic effect);t-butyl-phenylahistin exhibited its greatest potency against the PC-3tumor cell line, although the greatest efficacy was displayed againstthe HT-29 cell line; KPU-2 and KPU-35 were generally 10-40 fold morepotent than t-butyl-phenylahistin whereas the efficacy was similar forall three compounds in the different tumor cell lines; the HT-29, PC-3,MDA-MB-231 and NCI-H292 tumor cell lines all responded similarly to theNPI compounds, whereas the B16-F10 appeared to be somewhat lesssensitive. t-butyl-phenylahistin displayed a marked differential betweennormal fibroblasts and the tumor cell lines, with a ratio rangingfrom >20->100, except for the OVCAR-3 cell line. TABLE 12 Activity ofKPU-2, KPU-35 and t-butyl phenylahistin in the Tumor Panel Screen KPU-2KPU-35 t-butyl-phenylahistin Cell Line Mean SD n Mean SD n Mean SD nHT-29 Colon IC50 nM 9.8 2.4 4 8.2 2.0 4 420 473 3 % Cytotoxicity 82.55.3 4 81.3 4.0 4 88 0.2 3 PC-3 Prostate IC50 nM 13.4 0.7 4 13.2 2.5 4174 — 2 % Cytotoxicity 60.3 2.1 4 56.8 1.0 4 59.5 — 2 MDA-MB-231 BreastIC50 nM 13.8 1.9 3 9.7 4.2 4 387 — 2 % Cytotoxicity 56.7 7.2 3 59.3 5.64 65.5 — 2 NCI-H292 Lung IC50 nM 17.5 1.1 4 15.9 1.1 4 384 194 3 %Cytotoxicity 70.5 2.9 4 68.5 2.9 4 65 5 3 OVCAR-3 Ovary IC50 nM >20,000— 4 >20,000 — 4 >20,000 — 2 % Cytotoxicity 45.8 3.0 4 39 2.2 4 37 — 2B16-F10 Melanoma IC50 nM 37.1 26.3 4 32.3 19.9 4 736 650 3 %Cytotoxicity 71.8 2.5 4 72.0 2.2 4 74 2 3 CCD-27sk Fibroblast IC50 nM9.2 2.9 4 7.4 2.6 4 >20,000 — 2 % Cytotoxicity 64.3 2.4 4 60.8 1.9 4 45— 2

B). Studies in Drug Resistant Cell Lines

One of the major challenges in the use of chemotherapeutic agents inclinical oncology is the development of resistance to the drug effect bythe tumor cells. There are several mechanisms for the development ofresistance, each of which will have differential effects onchemotherapeutic drugs. These mechanisms include increased expression ofATP-dependent efflux pumps such as the P-glycoprotein encoded by MDR1 orthe multidrug-resistance associated protein 1 encoded by MRP1. Reduceddrug uptake, alteration of the drug's target, increasing repair ofdrug-induced DNA damage, alteration of the apoptotic pathway and theactivation of cytochrome P450 enzymes are other examples of mechanismsby which cancer cells become resistant to anticancer drugs. The selectedcompounds were studied in three different cell lines that exhibit twodifferent mechanisms of resistance; the overexpression of theP-glycoprotein and altered topoisomerase II activity.

1) Human Uterine Sarcoma Tumor Cell Line Pair: MES-SA (Taxol Sensitive)and MES-SA DX (Taxol Resistant).

This cell line expresses elevated mdr-1 mRNA and P-glycoprotein (anextrusion pump mechanism). Pretreatment with cyclosporin-A (CsA) blocksP-glycoprotein and reinstates activity in the resistant cell line forthose compounds for which the resistance is due to elevatedP-glycoprotein.

As can be seen from Table 13, KPU-2, and KPU-35 have the same potency inthe resistant cell line as in the sensitive line and the potency oft-butyl-phenylahistin was only slightly reduced. Cyclosporin A (CsA)pretreatment did not alter the potency of the selected compounds. Incontrast, taxol was virtually inactive in the MES-SA DX resistant cellline, whereas this compound was very potent in the sensitive cell line.CsA treatment restored the sensitivity to taxol of the MES-SA DX cellline. The MES-SA DX cell line also showed reduced susceptibility toetoposide (60 fold), doxorubicin (34 fold) and mitoxantrone (20 fold).

These data indicate that the effects of KPU-2, KPU-35 andt-butyl-phenylahistin are not susceptible to the taxol-relatedresistance mechanism (p-glycoprotein) in this cell line, and thatcross-resistance from taxol does not occur to these selected compoundsin this model. TABLE 13 Activity of KPU-2, KPU-35, t-butyl-phenylahistinand Taxol in MES-SA Taxol Sensitive and MES-SA DX Taxol Resistant HumanUterine Sarcoma Tumor Cell Lines MES-SA Sensitive MES-SA DX Resistant NoCsA Pretreat No CsA CsA Pretreat Compound CsA Ratio Ratio Ratio StudyIC50 nM IC50 nM No CsA IC50 nM MES-SA IC50 nM No CsA KPU-2 Study I 8.5 —— 10.5 1.2 — — Study II 19.4 27.4 1.4 21.7 1.1 37.8 1.74 KPU-35 Study I6.6 — 5.2 0.8 — — Study III 3.9 2.0 0.5 2.5 0.6 6.7 2.7 t-butyl-phenylahistin Study I 144 — — 825 5.7 — — Study III 122 162 1.3 694 4.3622 0.9 Taxol Study I 4.4 — — >20,000 >455 — — Study II 13.3 7.60.6 >>100 >>8 40 <<0.25 Study III 7.3 2.8 0.4 >24,000 >3000 2.0 <<0.0012) Human Acute Promyelocytic Leukemia Cell Line Pair: HL-60(Mitoxantrone-Sensitive) and HL-60/MX-2 (Mitoxantrone-Resistant)

This cell line is considered to have atypical drug resistance propertieswith altered topoisomerase II catalytic activity without overexpressionof P-glycoprotein.

As can be seen in Table 14, these results indicate that the potencies ofthe selected novel compounds are very similar in the sensitive andresistant HL-60 cell lines. In contrast, Mitoxantrone loses efficacy bya factor of 24-fold in the resistant HL-60/MX-2 cell line.

Thus, KPU-2, KPU-35 and t-butyl-phenylahistin are not susceptible to thesame resistance mechanisms as Mitoxantrone in this cell line, and thereis no cross-resistance from Mitoxantrone to these selected novelcompounds in this model. TABLE 14 Activity of KPU-2, KPU-35,t-butyl-phenylahistin and Mitoxantrone in the HL-60 Human AcutePromyelocytic Leukemia Tumor Sensitive and Resistant Cell Line PairHL-60 Resistant HL-60 Sensitive Ratio Compound IC50 nM IC50 nM toSensitive KPU-2 6.4 8.17 1.28 KPU-35 9.2 7.3 0.79 t-butyl-phenylahistin255 175 0.69 Mitoxantrone 202 4870 24.1

3). Human Breast Carcinoma Cell Line Pair: MCF-7 (Taxol Sensitive) andMCF-7/ADR (Taxol Resistant)

This study involved KPU-2 in comparison to taxol. KPU-2 demonstratedsimilar potencies in both the sensitive and resistant members of thiscell line pair. In contrast, taxol was virtually inactive in theresistant cell line whereas there was low nanomolar potency in thesensitive cell line (Table 15).

These studies confirm in a different human tumor cell line that taxolresistance does not transfer to KPU-2. TABLE 15 Activity of KPU-2 andTaxol in the MCF-7 Human Breast Carcinoma Sensitive and Resistant CellLine Pair MCF-7/ADR Resistant MCF-7 Sensitive Ratio Compound IC50 nM IC50 nM to Sensitive KPU-2 39.6 27.4 0.69 Taxol 2.6 >>100 >>38

C) Studies of the Mechanism of Action

1). Action on Microtubule Function

Human umbilical vein endothelial cells (HuVEC from Cambrex) were used inthis study, for evaluating the effects of KPU-2 andt-butyl-phenylahistin in comparison to colchicine and taxol on tubulinby staining for α-tubulin.

Thirty minutes exposure to KPU-2, t-butyl-phenylahistin or colchicine(all at 2 μM) induced microtubule depolymerization as was indicated bythe lack of intact microtubule structure in contrast to that observed inthe DMSO Control and cell membrane blebbing (a clear indication ofapoptosis) in the HuVEC cells, whereas taxol did not induce microtubuledepolymerization under these conditions. Colchicine is a knownmicrotubule depolymerizing agent whereas taxol is a tubulin stabilizingagent. Similar results were obtained when CCD-27sk cells were exposed toKPU-2 or colchicine.

2). Induction of Apoptosis

Apoptosis and its dysregulation play an important role in oncology; theselective induction of the programmed cell death cycle in tumor cells isthe goal of many chemotherapeutic drug discovery programs. Thisinduction of apoptosis can be demonstrated by different methodsincluding the characteristic cell membrane blebbing, DNA fragmentation,hyperphosphorylation of the antiapoptotic factor Bcl-2, activation ofthe caspase cascade and cleavage of poly (ADP ribose) polymerase (PARP).

The characteristic signs of apoptotic cell death include cell membraneblebbing, disruption of nuclei, cell shrinkage and condensation andfinally cell death, very distinctive from necrotic cell death. KPU-2induced the typical morphological changes associated with early stagesof apoptosis in human prostate tumor cells. A similar finding was alsoclear in the treatment of HuVEC cells with KPU-2.

3). DNA Fragmentation

A late stage characteristic of apoptosis is internucleosomal DNAcleavage that results in a distinctive ladder pattern that can bevisualized by gel electrophoresis. This approach was used to study theeffect of KPU-2 on DNA laddering in Jurkat cells (human T cell leukemialine) in comparison to halimide and dehydrophenylahistin (KPU-1). KPU-2induced DNA laddering at the 1 nM concentration whereas halimide andKPU-1 were much less potent.

4). Activation of the Caspase Cascade

Several enzymes in the caspase cascade are activated during apoptosis,including Caspase-3, -8 and -9. The activity of Caspase-3 was monitoredin Jurkat cells following treatment with KPU-2, KPU-35 andt-butyl-phenylahistin.

The results indicate that caspase-3 was activated in a dose-dependentmanner by treatment with all three compounds in a manner similar tohalimide. The caspase-3 activation occurred over a similar concentrationrange as for the IC50s for cytotoxicity in the Jurkat cell line (Table16). TABLE 16 Cytotoxicity of KPU-2, KPU-35 and t-butyl-phenylahistin inJurkat Cells Cytotoxicity Potency Efficacy NPI Compound IC50 nM % CellDeath KPU-2 11 94 KPU-35 5 93 t-butyl-phenylahistin 165 93 Mitoxantrone41 995). Cleavage of Poly(ADP-Ribose) Polymerase (PARP) in Jurkat Cells

In order to assess the ability of these compounds to induce apoptosis inJurkat cells, cleavage of poly(ADP-ribose) polymerase (PARP) wasmonitored. PARP is a 116 kDa nuclear protein that is one of the mainintracellular targets of Caspase-3. The cleavage of PARP generates astable 89 kDa product, and this process can be easily monitored bywestern blotting. Cleavage of PARP by caspases is one of the hallmarksof apoptosis, and as such serves as an excellent marker for thisprocess. KPU-2 at 100 nM induced cleavage of PARP in Jurkat cells 10hours after exposure of the cells to the compound. KPU-2 appeared to bemore active than either halimide or KPU-1.

6). Enhanced Vascular Permeability in HuVEC Cells

Compounds that depolymerize microtubules (e.g. combretastatinA-4-phosphate, ZD6126) have been shown to induce vascular collapse intumors in vivo. This vascular collapse is preceded by a rapid inductionof vascular cell permeability initially to electrolytes and soon afterto large molecules. The enhanced permeability of HuVEC cells to afluorescent-labeled dextran is used as a proxy assay for vascularcollapse.

KPU-2, KPU-35 and t-butyl-phenylahistin all rapidly (within 1 hour)induced significant HuVEC monolayer permeability, to an extent similarto colchicine. The microtubule stabilizing agent taxol was inactive inthis assay (FIG. 12).

7). Profile in a Broad Kinase Screen

KPU-2 was initially screened at a concentration of 10 μM in a panel of60 different kinases; the ATP concentration was 10 μM. Four kinases wereinhibited by greater than 50% in the primary screen and the IC50'sdetermined in secondary screening are presented in Table 17. All of theIC50 values are in the low micromolar range, which indicates thatinhibition of these kinases is not related to the low nanomolaractivities observed for tumor cell cytotoxicity. TABLE 17 Activity ofKPU-2 against Selected Kinases Kinase IC50 (μM) CDK1/Cyclin B (human)10.1 c-RAF (human) 8.9 JNK3 (rat) 6.8 Lyn (mouse) 11.1

EXAMPLE 9 In Vivo Pharmacology

Preliminary studies with KPU-2 were performed using the MX-1 (breast)and HT-29 (colon) xenograft models and the P-388 murine leukemia tumormodel, in the mouse. Other tumor models selected on the basis ofactivity in the in vitro tumor panel were the DU-145 (prostate), MCF-7(breast), and the A549 (lung) cell lines. The human pancreatic tumor(MiaPaCa-2) was also included. The novel compounds were studied asmonotherapy and in combination with a clinically-used chemotherapeuticagent. The doses of the selected novel compounds were determined fromthe acute tolerability testing (Maximally Tolerated Dose, MTD) and wereadjusted if necessary during each study. The doses of theclinically-used chemotherapeutic agents were selected on the basis ofhistorical studies.

KPU-2 was the first compound to be studied in these five tumor models.Following the initial results from this study, all three compounds werecompared in the HT-29 human colon tumor, the DU-145 human prostate andthe MCF-7 human breast tumor xenograft models.

The above models all use the subcutaneous xenograft implantationtechnique and are potentially subject to selective effects of a compoundon the subcutaneous vasculature producing a magnified (or apparent)antitumor activity. In order to circumvent this possibility, two othertumor models have been incorporated in the research. One of these is theobservation of lung metastases following the intravenous injection ofB16-F10 mouse melanoma tumor cells. The other model is the implantationof MDA-231 human breast tumor cells in the mouse mammary fat pad. Whilethis latter model is a xenograft model, the subcutaneous vasculaturedoes not play a role.

Methods

1). Xenograft Models

Animals used were (exceptions are indicated for individual studies):female nude mice (nu/nu) between 5 and 6 weeks of age (˜20 g, Harlan);group size was 9-10 mice per group unless otherwise indicated.

Cell lines used for tumor implantation were: HT-29 human colon tumor;MCF-7 human breast tumor; A549 human non small cell lung tumor;MiaPaCa-2 human pancreas tumor; DU-145 human prostate tumor.

Selected novel compounds were administered as monotherapy via theintraperitoneal (i.p.) route at the doses indicated for the individualstudy; for the combination studies the selected reference chemotherapyagents were injected 15-30 min prior to the compound.

Vehicles used in these studies were: 12.5% DMSO, 5% Cremaphor and 82.5%peanut oil for the selected novel compounds; (1:3) Polysorbate 80:13%ethanol for taxotere; (1:1) Cremaphor:ethanol for paclitaxel; for CPT-11each mL of solution contained 20 mg of irinotecan hydrochloride, 45 mgof sorbitol NF powder, and 0.9 mg of lactic acid, the pH being adjustedto 7.4 with NaOH or HCl. Saline dilutions are used to achieve theinjection concentrations used for the reference compounds.

HT-29 Human Colon Tumor Model

Animals were implanted subcutaneously (s.c.) by trocar with fragments ofHT-29 tumors harvested from s.c. growing tumors in nude mice hosts. Whenthe tumor size reached 5 mm×5 mm (about 10-17 days) the animals werematched into treatment and control groups. Mice were weighed twiceweekly and tumor measurements were obtained using calipers twice weekly,starting on Day 1. The tumor measurements were converted to estimated mgtumor weight using the formula (W²×L)/2. When the estimated tumor weightof the control group reached an average of 1000 mg the mice wereweighed, sacrificed and the tumor removed. The tumors were weighed andthe mean tumor weight per group was calculated and the tumor growthinhibition (TGI) was determined for each group (100% minus the change inthe mean treated tumor weight/the change in the mean control tumorweight'100.

In this model unless otherwise noted for the individual study, theselected novel compounds were injected intraperitoneally every third dayfor 15 days [1, 4, 8, 11 and 15 (q3d×5)]; CPT-11 was administeredintraperitoneally on days 1, 8 and 15 (qw×3).

MCF-7 Human Breast Tumor Model

Female nude mice (20 g) were implanted s.c. with 21-day release estrogen(0.25 mg) pellets 24 hours prior to the s.c. implantation with MCF-7tumor fragments (harvested from s.c. tumors in nude mice hosts). Thestudy then proceeded as described for the HT-29 model, using taxotere asthe standard chemotherapy agent.

In this model unless otherwise noted for the individual study, the novelcompounds were injected via the intraperitoneal route daily on Days 1-5,inclusive (qd×5); taxotere was administered intravenously on Days 1, 3and 5 (qod×3).

A549 Human Lung Tumor Model

Animals were implanted s.c. by trocar with fragments of A549 tumorsharvested from s.c. growing tumors in nude mice hosts. When the tumorsize reached 5 mm×5 mm (about 10-17 days) the animals were matched intotreatment and control groups. The rest of the study proceeded asdescribed for the HT-29 model, using taxotere and CPT-11 as the standardchemotherapy agents.

In this model unless otherwise noted for the individual study, thetested compounds were administered via the intraperitoneal route on aq3d×5 dose schedule for the CPT-11 combination or on a qd×5 dose regimenfor the combination with taxotere; CPT-11 was administered via theintraperitoneal route on a qw×3 schedule; taxotere was administeredintravenously on a qod×3 dose regimen.

MiaPaCa-2 Human Pancreas Tumor Model

Animals were implanted s.c. by trocar with fragments of MiaPaCa-2 tumorsharvested from s.c. growing tumors in nude mice hosts. When the tumorsize reached 5 mm×5 mm (about 10-17 days) the animals were matched intotreatment and control groups. The rest of the study proceeded asdescribed for the HT-29 model, using gemcitabine as the standardchemotherapy agent.

In this model unless otherwise noted for the individual study, testcompounds were administered every third day via the intraperitonealroute on Days 1, 4, 7, 10 and 15 (q3d×5); gemcitabine was administeredvia the intraperitoneal route on Days 1, 4, 7 and 10 (q3d×4).

DU-145 Human Prostate Tumor Model

Male mice were implanted s.c. by trocar with fragments of DU-145 tumorsharvested from s.c. growing tumors in nude male mice hosts. When thetumors reached 5 mm×5 mm ( at about 13-17 days) the animals were matchedinto treatment and control groups. The remainder of the study proceededas for the HT-29 model, using taxotere as the standard chemotherapyagent.

In this model unless otherwise noted for the individual study, testcompounds were administered via the intraperitoneal route on Days 1, 3,5, 8 and 11 (q3d×5); taxotere was administered intravenously on Days 1,3 and 5 (q2d×3).

2). Non Subcutaneous Implantation Tumor Models

The animals used were: female nude mice (nu/nu) (MDA-231 study) orB6D2F1 (B16-F10 studies) mice between 5 and 6 weeks of age (˜20 g,Harlan); group size was 10 mice per group unless otherwise indicated.

The cell lines used were: MDA-MB-231 human breast tumor and B16-F10murine melanoma cells.

NPI compounds were administered as monotherapy via the intraperitonealroute at the doses indicated for the individual study; for thecombination studies the selected reference chemotherapy agents wereinjected 15-30 min prior to the NPI compound.

MDA-231 Human Breast Tumor

Female nude mice were injected in the mammary fat pad with 2×10⁶ MDA-231cells harvested from in vitro cell culture. When the tumor size reached5 mm×5 mm (about 14-28 days) the animals were matched into treatment andcontrol groups. The study then proceeded as described for the HT-29model, using paclitaxel as the standard chemotherapy agent.

In this model unless otherwise noted for the individual study, the testcompounds were administered via the intraperitoneal route on Days 1, 4,8, 11 and 15 (q3d×5); paclitaxel was administered via theintraperitoneal route on Days 1-5 (qd×5). B16-F10 MetastaticMurineMelanoma Model

Mice received B16-F10 cells (prepared from an in vitro cell culture ofB16-F10 cells) by the iv route on Day 0. On Day 1 mice were randomizedinto treatment and control groups and treatment commenced. Mice wereweighed twice weekly, starting on Day 1. All mice are sacrificed on Day16, the lungs removed, weighed and the surface colonies counted. Resultsare expressed as mean colonies of treated mice/mean colonies of controlmice (T/C)×100%). The metastasis growth inhibition (MGI) is this numbersubtracted from 100%. Paclitaxel was the standard chemotherapy agentused in this study.

In this model unless otherwise noted for the individual study, the testcompounds were administered via the intraperitoneal route on Days 1-5(qd×5); paclitaxel was administered intravenously on Days 1-5(qd×5).

When appropriate (n 3), results are presented as means±SEM. Statisticalanalysis of studies with several groups was performed using ANOVA withNeuman-Keuls post test, unless otherwise indicated. A one-tailed t-testwas also used based on the hypothesis that the compound or drug, or thecombination, would reduce tumor growth.

Results Studies in the HT-29 Human Colon Tumor Xenograft Model

1. In Vivo Evaluation of KPU-2±CPT-11 in the HT-29 Human Colon TumorXenograft Model

This study assessed changes in dosage strength and dosing regimen forKPU-2 alone and in combination with a relevant chemotherapeutic CPT-11in the HT-29 model.

KPU-2 was administered at doses of 7.5 mg/kg ip daily for five days(qd×5), 3.75 mg/kg ip bid for five days, 7.5 mg/kg ip every second dayfor 10 days (qod×5) and 7.5 mg/kg ip every third day for 15 days(q3d×5). The combination of CTP-11 with NPI-2358 at a dose of 7.5 mg/kgip q3d×5 resulted in a significantly greater effect than for eithercompound alone, which lasted for the duration of the study (FIG. 13).These observations during the in-life portion of the study wereconfirmed by the mean group final tumor weights at autopsy for whichonly the combination group exhibited a statistically significant lowertumor weight than controls. In addition the difference between the meantumor weights of the combination therapy and CPT-11 monotherapy groupswas statistically significant (FIG. 14). When the individual final tumorweights at autopsy are examined the greater effect of cotherapy is clear(FIG. 14). The TGI of cotherapy was 78% as compared to 38.9% for CPT-11alone. The TGI for the combined therapy group exceeds the NCI criterionof 58% for a positive result.

2. Study of KPU-2±Standard Chemotherapy vs. Five Human Tumor XenograftModels

This study consists of five different arms, each with its own protocol,timing, dosing regimen and reference compound. Each arm will beconsidered within the presentation of the particular tumor model.

The aim of the HT-29 arm of the study was to investigate a slightlyhigher dose of KPU-2 (10 mg/kg ip q3d×5) in the HT-29 human colon tumorxenograft model as compared to those used in the study described above,in which a marked synergy was observed between KPU-2 (7.5 mg/kg ipq3d×5) and CPT-11 (100 mg/kg ip qw×3).

As can be observed in FIG. 15, the combination of KPU-2 and CPT-11 inthis model resulted in a marked synergy in the inhibition of tumorgrowth, with the tumor growth being almost completely inhibited up toTreatment Day 29 in the combination therapy group. The combined therapymaintained efficacy and the estimated tumor growth for this group wassignificantly lower than for either monotherapy group. Accordingly,administration of KPU-2 and CPT-11 inhibited tumor growth and is aneffective anti-tumor treatment.

The observations of the in-life portion of the study (estimated tumorgrowth, FIG. 15) are supported by measurement of the weights of thetumors excised at autopsy (FIG. 16). The tumor weights for thecombination group was significantly less than the Controls (p<0.01), aswere the tumor weights for CPT-11 alone (p<0.05).

When the individual final tumor weights are considered (FIG. 16), thetumor size for the combination group was generally smaller than for theother treated or control groups. The TGI of the combination group was65.8%, indicating a positive effect by the NCI criterion, whilemonotherapy did not reach the NCI criterion of TGI>58%.

3. Study of Activity of KPU-2, KPU-35 and t-butyl-phenyalhistin in theHT-29 Human Colon Tumor Xenograft Study

The results of this study are presented in FIG. 17 and Table 18. Thecombination therapy groups all indicated a marked synergy between thenovel compounds and CPT-11. The individual tumor weights demonstrate theeffectiveness of the cotherapy treatment (FIG. 18). In each case the TGIfor the combination group surpasses the NCI criterion for a positiveeffect, whereas the TGI for CPT-11 monotherapy did not reach this level.TABLE 18 Summary of Studies Performed in the HT-29 Human Colon TumorModel NPI- Chemotherapeutic Combination Study Description Compound AgentExceed NCI Number Number, Result Name, Result Results Criterion StatusEndpoint mg/kg ip TGI % Dose TGI % TGI % (TGI 58%) Comments 2164 TGIKPU-2 CPT-11 39* 78**;# Combination Synergy 7.5 qdx5 No Effect 100 ip7.5 q3dx5 No Effect qwx3 2288 TGI KPU-2 No Effect CPT-11 36.5* 65.8**Combination Synergy 10 7.5 100 ip See Text q3dx5 qwx3 2139 TGI KPU-2 NoEffect CPT-11 32.7 80.7**,# Combination Synergy 7.5 100 ip q3dx5 qwx32139 TGI KPU-35 No Effect CPT-11 32.7 83.3**,## Combination Synergy 107.5 100 ip 1+ Day 13 q3dx5 qwx3 1+ Day 27 2139 TGI t-butyl- No EffectCPT-11 32.7 77.7*,# Combination Synergy phenylahistin 100 ip 30 qwx3q3dx5*p < 0.05 vs Control;**p < 0.01 vs Control;#p < 0.05 vs CPT-11 Alone;##p < 0.01 vs CPT-11 Alone;+ = Number of Deaths4. Summary of the Effects of KPU-2,KPU-35 and t-butyl-phenylahistin inCombination with CPT-11 in the HT-29 Human Colon Tumor Xenograft Model

When combined with CPT-11, KPU-2 enhanced the effect of CPT-11, thestandard chemotherapeutic agent, to a level well in excess of the NCIcriterion of a TGI 58% for a positive effect. The results generated inthe three studies are very comparable for both the in-life observations(FIG. 19) and for the weights of the tumors excised at autopsy (FIG.20).

Studies in the DU-145 Human Prostate Tumor Xenograft Model

Two studies have been completed with this model: the first studyinvolved KPU-2 alone and in combination with taxotere; the second studycompared KPU-2, KPU-35 and t-butyl-phenyalhistin alone and incombination with taxotere.

1. Effect of KPU-2 in Combination with Taxotere in the DU-145 HumanProstate Tumor Xenograft Model

As can be seen from the data obtained during the in-life portion of thisstudy (FIG. 21), the most effective treatment of the DU-145 humanprostate tumor was the combined therapy of KPU-2 plus taxotere. Thetreatment effect was most pronounced at the beginning of the study andappeared to be reduced as the study progressed. From treatment Days20-27, the combination therapy did provide an apparent TGI that exceededthe NCI criterion (TGI 58%), and the estimated tumor weight of thecombined therapy was significantly less than for either monotherapy.

2. Activity of KPU-2, KPU-35 and t-butyl-phenylahistin Alone or inCombination with Taxotere in the DU-145 Human Prostate Xenograft Model

Based on the data obtained with KPU-2 in combination with taxotere inthe Study described above a second study comparing KPU-2 to KPU-35 andt-butyl-phenylahistin alone and in combination with taxotere wasinitiated.

The observations made during the in-life portion of this study indicatethat the combination of either KPU-2 or KPU-35 with taxotere has agreater reduction on tumor growth than for taxotere alone (FIG. 22). Thetumor growth was almost completely blocked by KPU-35 in combination withtaxotere.

The excised tumor weights at autopsy confirmed the observations madeduring the in-life segment of the study. The combination of either KPU-2(FIG. 23) or KPU-35 (FIG. 24) with taxotere was significantly moreeffective than taxotere alone in blocking tumor growth. In the case ofKPU-35, three of ten mice showed evidence for tumor shrinkage. The tumorgrowth inhibition indices indicated a marked inhibition of tumor growthfor KPU-2 (group mean=74.1%) and an almost total block for KPU-35 (groupmean=92.5%). Taxotere alone did not reach the NCI established criterionfor a positive effect (TGA 58%).

5. Studies in the MCF-7 Human Breast Tumor Xenograft Model

This study compared the effects of KPU-2, KPU-35 andt-butyl-phenylahistin in the MCF-7 human breast tumor xenograft model.The doses of the compounds were administered on Days 1, 2, 3, 4, and 7;Taxotere was administered on Days 1, 3 and 7.

The selected novel compounds have early onset, statistically significanteffects when used in combination with taxotere in this model, apparentlyalmost completely blocking estimated tumor growth (FIG. 25). Of thethree compounds, KPU-2 appeared to be the most effective, witht-butyl-phenylahistin also exhibiting a significant potentiation oftaxotere.

6. Studies in the A549 Human Non Small Cell Lung Tumor Xenograft Model

The in-life observations during this study (FIG. 26) indicated that thecombination of KPU-2 (7.5 mg/kg ip, qd×5) with taxotere resulted in amarked inhibition of tumor growth as compared to the Control or eithermonotherapy group. This was confirmed by the autopsy tumor weights, asthe mean of the cotherapy group was significantly less than that oftaxotere alone or the Control group (FIG. 27). The cotherapy group tumorweights form a cluster of low tumor weights, indicating the consistencyof the effect.

When the tumor growth index is calculated, the cotherapy group had a TGIof 74.4% as compared to the control group well in excess of the NCIcriterion for a positive effect (TGI 58%). Taxotere alone had a TGI of26.1%.

7. Studies in the MDA-231 Human Breast Tumor Orthotopic Xenograft Model

This model involves the placement of the human tumor tissue into themouse mammary fat pad, a surrogate of the natural environment. In thismanner the possibility of a positive effect due to a specific action onthe subcutaneous vascular bed is avoided. This study compared the effectof KPU-2 (7.5 mg/kg ip, q3d×5) alone and in combination with paclitaxel(16 mg/kg ip, qd×5).

Three weeks into the study there was a significant inhibition of tumorgrowth in the combination therapy group, a highly significant effect.This effect appeared to be more marked than for taxotere alone (FIG.28).

8. Studies in the Murine Melanoma B16 F10 Metastatic Tumor Model

This study examined the effect of KPU-2, KPU-35 andt-butyl-phenylahistin alone and in combination with paclitaxel on thenumber of metastases appearing on the surface of the lung 16 days afterthe intravenous injection of B16 F10 melanoma cells to the mouse. Thismodel is not a xenograft model; however, it does not involve a highdegree of vascularization into the tumor mass.

In this model the most effective treatment was KPU-2 alone (FIG. 29),having a mean metastases count about 10% less than that for paclitaxel(MGIs of 41.6% and 35.0%, respectively). While this study does notitself establish that combination therapy is more effective thanmonotherapy, it does indicate that KPU-2, KPU-35 andt-butyl-phenylahistin are most effective in highly vascularized tumors.

EXAMPLE 10 Assays For Activity Against Pathogenic Fungi

Comparative activity of a dehydrophenylahistin or its analog against apathogenic fungus, relative to known antifungal compounds recited above,for use in determining the dehydrophenylahistin or its analog's AF/ISvalue is measured directly against the fungal organism, e.g. bymicrotiter plate adaptation of the NCCLS broth macrodilution methoddescribed in Diagn Micro and Infect Diseases 21:129-133 (1995).Antifungal activity can also be determined in whole-animal models offungal infection. For instance, one may employ the steroid-treated mousemodel of pulmonary mucormycosis (Goldaill, L. Z. & Sugar, A. M. 1994 JAntimicrob Chemother 33:369-372). By way of illustration, in suchstudies, a number of animals are given no dehydrophenylahistin or itsanalog, various doses of dehydrophenylahistin or its analog (and/orcombinations with one or more other antifungal agents), or a positivecontrol (e.g. Amphotericin B), respectively, beginning before, at thetime of, or subsequent to infection with the fungus. Animals may betreated once every 24 hours with the selected dose ofdehydrophenylahistin or its analog, positive control, or vehicle only.Treatment is continued for a predetermined number of days, e.g. up toten days. Animals are observed for some time after the treatment period,e.g. for a total of three weeks, with mortality being assessed daily.Models can involve systemic, pulmonary, vaginal and other models ofinfection with or without other treatments (e.g. treatment withsteroids) designed to mimic a human subject susceptible to infection.

To further illustrate, one method for determining the in vivotherapeutic efficacies (ED₅₀, e.g. expressed in mg dehydrophenylahistinor its analog/kg subject), is a rodent model system. For example, amouse is infected with the fungal pathogen such as by intravenousinfection with approximately 10 times the 50% lethal dose of thepathogen (10⁶ C. albicans cells/mouse). Immediately after the fungalinfection, dehydrophenylahistin compounds are given to the mouse at apredetermined dosed volume. The ED₅₀ is calculated by the method of Vander Waerden (Arch Exp Pathol Pharmakol 195:389-412, 1940) from thesurvival rate recorded on 20th day post-infection. Generally, untreatedcontrol animals die 7 to 13 days post-infection.

In another illustrative embodimemt, C. albicans Wisconsin (C43) and C.tropicalis (C112), grown on Sabouraud dextrose agar (SDA) slants for 48h at 28° C., are suspended in saline and adjusted to 46% transmission at550 nm on a spectrophotometer. The inoculum is further adjusted byhemacytometer and confirmed by plate counts to be approximately 1 or5×10⁷ CFU/ml. CF-1 mice are infected by injection 1 or 5×10⁶ CFU intothe tail vein. Antifungal agents are administered intravenously orsubcutaneously in ethanol:water (10:90), 4 h post infection and oncedaily thereafter for 3 or 4 more days. Survival is monitored daily. TheED₅₀ can be defined as that dose which allows for 50% survival of mice.

EXAMPLE 11 Evaluating Antimicotic Activity

Benzimidazoles and griseofulvin are anti-tubulin agents capable ofbinding to fungal microtubules. Once bound, these compounds interferewith cell division and intracellular transport in sensitive organisms,resulting in cell death. Commercially, benzimidazoles are used asfungicidal agents in veterinary medicine and plant disease control. Awide variety of fungal species, including Botrytis cinerea, Beauveriabassiana, Helminthosporium solani, Saccharomyces cerevisiae andAspergillus are susceptible to these molecules. Toxicity concerns andincreasing drug resistance, however, have negatively impacted theirusage. Griseofilvin is used clinically to treat ringworm infections ofthe skin, hair and nails, caused by Trichophyton sp., Microsporum sp.,and Epidermophyton floccosum. Its antifungal spectrum, however, isrestricted to this class of fungal organisms. Genotoxicity is also asignificant side effect. Terbinafine, while an alternative first-linetreatment, is more costly. Further, clinical resistance recently hasbeen observed in Trichophyton rubrum (the major causative agent for alldermatophyte infections).

In Candida albicans, microtubule/microfilament formation is affectedwhere cells are exposed to the microtubule inhibitors nocodazole andchloropropham. These results further validate the exploration ofcytoskeleton inhibitors as effective antimycotic agents. Accordingly,several of the compounds disclosed herein were evaluated for antimycoticactivity.

Specifically, disclosed compounds were evaluated alongside commerciallyavailable microtubulin inhibitors as well as recognized antifungalagents. The test compounds and controls used in this study:(−)-Phenylahistin, KPU-1, KPU-2, KPU-11 and KPU-17, KPU-35, t-butylphenylahistin, Colchicine (commercial microtubulin inhibitor testedversus 3 Candida isolates), Benomyl (commercial microtubulin inhibitortested versus 3 Candida isolates), Griseofulvin (commercial microtubulininhibitor and antibiotic control for testing versus 6 dermatophyteisolates), Amphotericin B (antibiotic control for testing versus 3Candida isolates), Itraconazole (antibiotic control for testing versus 2Aspergillus isolates).

Microorganisms against which these compounds were tested included:Candida albicans, Candida glabrata, Aspergillus fumigatus, Trichophytonrubrum, Trichophyton mentagrophytes, Epidermophyton floccosum. With theexception of Candida glabrata (one isolate), two isolates of eachspecies were tested.

Antifungal susceptibility testing was accomplished according to themethods outlined in the National Committee for Clinical LaboratoryStandards, M38-A “Reference Method for Broth Dilution AntifungalSusceptibility Testing of Conidium-Forming Filamentous Fungi; ApprovedStandard.” This includes testing in RPMI-1640 with glutamine and withoutbicarbonate, an inoculum size of 0.4-5×10⁴, and incubation at 30 or 35°C. for 48 hours. The minimum inhibitory concentration (MIC) was definedas the lowest concentration that resulted in an 80% reduction inturbidity as compared to a drug-free control tube. Drug concentrationswere 0.03-16 μg/ml for the investigational compounds, 0.015-8 μg/ml foritraconazole and griseofulvin.

The minimum inhibitory concentration (IC) at which a compound preventedthe growth of the target microorganism was assessed according to themodified version of the NCCLS protocol. Minimum inhibitoryconcentrations (MIC) were determined at the first 24-hour interval wheregrowth could be determined in the drug-free control tube. The definedMIC was the lowest concentration that exhibited an 80% reduction inturbidity as compared to the growth control. The minimum lethalconcentration (MLC) was determined by plating 0.1 μl from the MICconcentration and each concentration above the MIC. The MLC was calledat the first concentration that exhibited five or fewer colonies offungal growth representing a 99.95% kill. When a MIC was obtained, aminimum fungicidal concentration (MFC) was determined to assess thefungistatic/fungicidal nature of the compound. This procedure entailsdiluting drug-treated cell samples (removed from test wells containingcompound at and above the MIC) to compound concentrations significantlybelow the inhibitory concentration and depositing them on agar plates.The compound is scored as fungistatic if the cells are able to resumegrowth and fungicidal if no regrowth is possible because the compoundhad killed the organisms.

Compounds disclosed herein were shown to be effective against twoTrichophyton species. T. rubrum is the principal causative agent forhuman dermatophytic infections, and would be the key organism to targetin the development of a clinical agent.

Compounds KPU-2, KPU-11 and KPU-17, KPU-35 & t-butylphenylahistin wereequivalent in potency or in some cases more potent than griseofulvin, acurrent, standard pharmaceutical agent used for treating dermatophyticinfections.

Compounds (−)-Phenylahistin and KPU-1 were significantly less potentthan the other compounds when tested versus T. rubrum and weaker butmore comparable to the other versus the sensitive T. mentagrophytesisolate.

In those instances when an MFC could be determined, the results indicatethat these compounds are fungistatic in nature (see Tables 19 and 20).TABLE 19 Antifungal Activity of Dehydrophenylahistins and AnalogsThereof MICs and MFCs, μg/ml A. fumigatus A. fumigatus C. albicans 90028C. albicans 10231 C. glabrata isolate #1 isolate #2 Compound MIC MFC MICMFC MIC MFC MIC MFC MIC MFC (−)-Phenylahistin  >70 ND**  >70* ND >70ND >16 ND >16 ND KPU-1  >68* ND  >68 ND >68 ND >16 ND >16 ND KPU-2  >32ND  >32 ND >32 ND >16 ND >16 ND KPU-11 and KPU-  >32 ND  >32 ND >32ND >16 ND 0.06 >16 17 KPU-35  >32 ND  >32 ND >32 ND >16 ND <0.03 0.125t-butyl phenylahistin  >32 ND  >32 ND >32 ND >16 ND <0.03 0.125amphotericin B    0.5 0.5    0.5 0.5 1 1 ND ND ND ND griseofulvin ND NDND ND ND ND ND ND 0.5 ND itraconazole ND ND ND ND ND ND 1 ND ND NDcolchicine >128 ND >128 ND >128 ND ND ND ND ND benomyl    64 >512   64 >512 64 >512 ND ND ND ND

TABLE 20 Antifungal Activity of Dehydrophenylahistins and AnalogsThereof MICs and MFCs, μg/ml T. rubrum T. rubrum T. mentagrophytes T.mentagrophytes E. floccosum E. floccosum isolate #1 isolate #2 isolate#1 isolate #2 isolate #1 isolate #2 Compound MIC MFC MIC MFC MIC MFC MICMFC MIC MFC MFC NPI2350 >16 ND 0.16 >16 16 >16 >16 ND >16 ND >16 NDNPI2352 >16 ND 0.25 >16 4 >16 >16 ND >16 ND >16 ND NPI2358 >16 ND <0.030.125 2 >16 >16 ND >16 ND >16 ND NPI2362 0.06 >16 <0.03 <0.03 1 >16 >16ND >16 ND >16 ND NPI2386 <0.03 0.125 <0.03 0.06 1 >16 >16 ND >16 ND >16ND NPI2460 <0.03 0.125 <0.03 <0.03 4 >16 >16 ND >16 ND >16 NDamphotericin B ND ND ND ND ND ND ND ND ND ND ND ND griseofulvin 0.5 ND<0.015 ND 1 ND 2 ND 2 ND 4 ND itraconazole ND ND ND ND ND ND ND ND ND NDND ND colchicine ND ND ND ND ND ND ND ND ND ND ND ND benomyl ND ND ND NDND ND ND ND ND ND ND ND

The examples described above are set forth solely to assist in theunderstanding of the invention. Thus, those skilled in the art willappreciate that the disclosed methods and compounds encompass and mayotherwise provide further derivatives of dehydrophenylahistins.

One skilled in the art would readily appreciate that the presentinvention is well adapted to obtain, for example, the ends andadvantages mentioned, as well as others inherent. The methods andprocedures described herein are presently representative of preferredembodiments and are exemplary and are not intended as limitations on thescope of the invention. Changes therein and other uses will occur tothose skilled in the art which are encompassed within the spirit of theinvention.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.

As noted above, all patents and publications mentioned in thespecification are indicative of the levels of those skilled in the artto which the invention pertains. All patents and publications are herebyincorporated by reference herein to the extent allowable by law, suchthat each individual patent and publication may be treated asspecifically and individually indicated to be incorporated by reference.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and there is no intention that in the use of such terms andexpressions indicates the exclusion of equivalents of the features shownand described or portions thereof. It is recognized that variousmodifications are possible within the scope of the invention. Thus, itshould be understood that although the present invention has beenspecifically disclosed by preferred embodiments and optional features,modification and variation of the concepts herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be falling within the scope of theinvention.

1. A method for the synthetic preparation of a compound having thestructure of Formula (I):

wherein R₁, R₄, and R₆, are each separately selected from the groupconsisting of a hydrogen atom, a halogen atom, and saturated C₁-C₂₄alkyl, unsaturated C₁-C₂₄ alkenyl, cycloalkyl, cycloalkenyl, alkoxy,cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl,amino, substituted amino, nitro, azido, substituted nitro, phenyl, andsubstituted phenyl groups, hydroxy, carboxy, —CO—O—R₇, cyano, alkylthio,halogenated alkyl including polyhalogenated alkyl, halogenated carbonyl,and carbonyl —CCO—R₇, wherein R₇ is selected from a hydrogen atom, ahalogen atom, and saturated C₁-C₂₄ alkyl, unsaturated C₁-C₂₄ alkenyl,cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl,heteroaryl, substituted heteroaryl, amino, substituted amino, nitro,azido, substituted nitro, phenyl, and substituted phenyl groups; R₁′ andR₁″ are each independently selected from the group consisting of ahydrogen atom, a halogen atom, and saturated C₁-C₂₄ alkyl, unsaturatedC₁-C₂₄ alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, amino, substitutedamino, nitro, azido, substituted nitro, phenyl, and substituted phenylgroups, hydroxy, carboxy, —CO—O—R₇, cyano, alkylthio, halogenated alkylincluding polyhalogenated alkyl, halogenated carbonyl, and carbonyl—CCO—R₇, wherein R₇ is selected from a hydrogen atom, a halogen atom,and saturated C₁-C₂₄ alkyl, unsaturated C₁-C₂₄ alkenyl, cycloalkyl,cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl,substituted heteroaryl, amino, substituted amino, nitro, azido,substituted nitro, phenyl, and substituted phenyl groups; R, R₁′ and R₁″are either covalently bound to one another or are not covalently boundto one another; R₂, R₃, and R₅ are each separately selected from thegroup consisting of a hydrogen atom, a halogen atom, and saturatedC₁-C₁₂ alkyl, unsaturated C₁-C₁₂ alkenyl, acyl, cycloalkyl, alkoxy,cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl,amino, substituted amino, nitro, and substituted nitro groups, sulfonyland substituted sulfonyl groups; X₁ and X₂ are separately selected fromthe group consisting of an oxygen atom, a nitrogen atom, and a sulfuratom, each either unsubstituted or substituted with a R₅ group, asdefined above; Y is selected from the group consisting of a nitrogenatom, a nitrogen atom substituted with R₅, an oxygen atom, a sulfuratom, a oxidized sulfur atom, a methylene group and a substitutedmethylene group; n is an integer equal to zero, one or two; Z, for eachseparate n, if non-zero, and Z₁, Z₂, Z₃ and Z₄ are each separatelyselected from a carbon atom, a sulfur atom, a nitrogen atom or an oxygenatom; and the dashed bonds may be either single or double bonds; saidmethod comprising: reacting a diacyldiketopiperazine with a firstaldehyde to produce an intermediate; and reacting said intermediate witha second aldehyde to produce said compound, wherein said first aldehydeand said second aldehydes are selected from the group consisting of anoxazolecarboxaldeyhyde, imidazolecarboxaldehyde, a benzaldehyde,imidazolecarboxaldehyde derivatives, and benzaldehyde derivatives,thereby forming the compound.
 2. The method according to claim 1,wherein said first aldehyde is an imidazolecarboxaldehyde.
 3. The methodaccording to claim 1, wherein said second aldehyde is a benzaldehyde. 4.The method according to claim 1, wherein each of R₂, R₃, R₅ and R₆ is ahydrogen atom.
 5. The method according to claim 1, wherein each of X₁and X₂ is an oxygen atom.
 6. The method according to claim 1, wherein R₄is a saturated C₁-C₁₂ alkyl.
 7. The method according to claim 6, whereinsaid saturated C₁-C₁₂ alkyl is a tertiary butyl group.
 8. The methodaccording to claim 1, wherein R₁ comprises a substituted phenyl.
 9. Themethod according to claim 8, wherein said substituted phenyl group ismethoxybenzene.
 10. The method according to claim 1, wherein said firstaldehyde is a benzaldehyde.
 11. The method according to claim 1, whereinsaid second aldehyde is an imidazolecarboxaldehyde.
 12. The methodaccording to claim 1, wherein n is equal to zero or one.
 13. The methodaccording to claim 1, wherein n is equal to one.
 14. The methodaccording to claim 1, wherein n is equal to one and Z, Z₁, Z₂, Z₃ and Z₄are each a carbon atom.
 15. A compound having the structure of Formula(I):

wherein R₁, R₄, and R₆, are each separately selected from the groupconsisting of a hydrogen atom, a halogen atom, and saturated C₁-C₂₄alkyl, unsaturated C₁-C₂₄ alkenyl, cycloalkyl, cycloalkenyl, alkoxy,cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl,amino, substituted amino, nitro, azido, substituted nitro, phenyl, andsubstituted phenyl groups, hydroxy, carboxy, —CO—O—R₇, cyano, alkylthio,halogenated alkyl including polyhalogenated alkyl, halogenated carbonyl,and carbonyl —CCO—R₇, wherein R₇ is selected from a hydrogen atom, ahalogen atom, and saturated C₁-C₂₄ alkyl, unsaturated C₁-C₂₄ alkenyl,cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl,heteroaryl, substituted heteroaryl, amino, substituted amino, nitro,azido, substituted nitro, phenyl, and substituted phenyl groups; R₁′ andR₁″ are each independently selected from the group consisting of ahydrogen atom, a halogen atom, and saturated C₁-C₂₄ alkyl, unsaturatedC₁-C₂₄ alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl,substituted aryl, heteroaryl, substituted heteroaryl, amino, substitutedamino, nitro, azido, substituted nitro, phenyl, and substituted phenylgroups, hydroxy, carboxy, —CO—O—R₇, cyano, alkylthio, halogenated alkylincluding polyhalogenated alkyl, halogenated carbonyl, and carbonyl—CCO—R₇, wherein R₇ is selected from a hydrogen atom, a halogen atom,and saturated C₁-C₂₄ alkyl, unsaturated C₁-C₂₄ alkenyl, cycloalkyl,cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl,substituted heteroaryl, amino, substituted amino, nitro, azido,substituted nitro, phenyl, and substituted phenyl groups; R, R₁′ and R₁″are either covalently bound to one another or are not covalently boundto one another; R₂, R₃, and R₅ are each separately selected from thegroup consisting of a hydrogen atom, a halogen atom, and saturatedC₁-C₁₂ alkyl, unsaturated C₁-C₁₂ alkenyl, acyl, cycloalkyl, alkoxy,cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl,amino, substituted amino, nitro, and substituted nitro groups, sulfonyland substituted sulfonyl groups; X₁ and X₂ are separately selected fromthe group consisting of an oxygen atom, a nitrogen atom, and a sulfuratom, each either unsubstituted or substituted with a R₅ group, asdefined above; Y is selected from the group consisting of a nitrogenatom, a nitrogen atom substituted with R₅, an oxygen atom, a sulfuratom, a oxidized sulfur atom, a methylene group and a substitutedmethylene group; n is an integer equal to zero, one or two; Z, for eachseparate n, if non-zero, and Z₁, Z₂, Z₃ and Z₄ are each separatelyselected from a carbon atom, a sulfur atom, a nitrogen atom or an oxygenatom; and the dashed bonds may be either single or double bonds; withthe proviso that, in a particular compound, if R₁, R₁′, R₂, R₃, R₄ andR₅ are each a hydrogen atom, then it is not true that X₁ and X₂ are eachan oxygen atom and R₆ is either 3,3-dimethylbutyl-1-ene or a hydrogenatom.
 16. The compound of claim 15, wherein each of R₂, R₃, R₅ and R₆ isa hydrogen atom.
 17. The compound of claim 15, wherein each of X₁ and X₂is an oxygen atom.
 18. The compound of claim 15, wherein R₄ is asaturated C₁-C₁₂ alkyl.
 19. The compound of claim 15, wherein thesaturated C₁-C₁₂ alkyl is a tertiary butyl group.
 20. The compound of toclaim 15, wherein R₁ is a substituted phenyl group.
 21. The compound ofclaim 20, wherein the substituted phenyl group is methoxybenzene. 22.The compound according to claim 15, wherein n is equal to zero or one.23. The compound according to claim 15, wherein n is equal to one. 24.The compound according to claim 15, wherein n is equal to one and Z, Z₁,Z₂, Z₃ and Z₄ are each a carbon atom.
 25. The compound of claim 15,wherein said compound is selected from the group consisting of: KPU-2,KPU-11, KPU-35, KPU-66, KPU-80, KPU-81, KPU-90 andt-butyl-phenylahistin.
 26. A pharmaceutical composition, comprising thecompound of claim 15 and a pharmaceutically acceptable carrier.
 27. Thepharmaceutical composition of claim 26, wherein said compound isselected from the group consisting of: KPU-11, KPU-80, KPU-81 andKPU-90.
 28. The pharmaceutical composition of claim 26, wherein saidcompound has a cytotoxic activity.
 29. The pharmaceutical composition ofclaim 26, wherein said compound is a cell-cycle inhibitor.
 30. A methodfor the treatment of a disease state in a mammal, comprisingadministering to the mammal a pharmaceutically effective amount of thecomposition of claim
 26. 31. The method of claim 30, wherein saiddisease state is neoplastic.
 32. The method of claim 30, wherein saiddisease state is a fungal infection.
 33. A pharmaceutical compositionfor treating or preventing fungal infection comprising an antifungallyeffective amount of a compound of claim 15 together with apharmaceutically acceptable carrier therefor.
 34. The composition ofclaim 33, wherein said compound is selected from the group consistingof: KPU-2, KPU-11, KPU-35, KPU-66, KPU-80, KPU-81, KPU-90 andt-butyl-phenylahistin.
 35. A method of treating and/or preventing atleast one fungal infection in a mammal afflicted with at least onefungal infection which comprises administering an antifungally effectiveamount of a compound of claim 15 sufficient for such treating orpreventing.
 36. The method of claim 35, wherein said compound isselected from the group consisting of: KPU-2, KPU-11, KPU-35, KPU-66,KPU-80, KPU-81, KPU-90 and t-butyl-phenylahistin.
 37. A pharmaceuticalcomposition for treating or preventing tumor comprising anpharmaceutically effective amount of a compound of claim 15 togetherwith a pharmaceutically acceptable carrier therefor.
 38. The method ofclaim 37, wherein said compound is selected from the group consistingof: KPU-2, KPU-11, KPU-35, KPU-66, KPU-80, KPU-81, KPU-90 andt-butyl-phenylahistin.
 39. A method of treating and/or preventing cancerin a mammal afflicted with cancer which comprises administering anantineoplastic amount of a compound of claim 15 sufficient for suchtreating or preventing.
 40. The method of claim 39, wherein saidcompound is selected from the group consisting of: KPU-2, KPU-11,KPU-35, KPU-66, KPU-80, KPU-81, KPU-90 and t-butyl-phenylahistin.